Methods, kits and compositions pertaining to the suppression of the detectable probe binding to randomly distributed repeat sequences in genomic nucleic acid

ABSTRACT

This invention is directed to methods, kits, non-nucleotide probes as well as other compositions pertaining to the suppression of binding of detectable nucleic acid probes to undesired nucleotide sequences of genomic nucleic acid in assays designed to determine target genomic nucleic acid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/368,732, filed Feb. 8, 2012, now U.S. Pat. No. 8,754,191, which is acontinuation of U.S. application Ser. No. 12/619,664, filed Nov. 16,2009, now abandoned, which is a continuation of U.S. application Ser.No. 10/255,434, filed Sep. 24, 2002, now abandoned, and claims thebenefit of U.S. application No. 60/324,499, filed on Sep. 24, 2001, thedisclosures of each of which is herein incorporated by reference in itsentirety.

BACKGROUND

1. Field of the Invention

This invention pertains to the field of molecular cytogenetics and morespecifically this invention pertains methods, kits and compositionsbeing used to suppress the binding of detectable nucleic acid probes toundesired sequences, such as randomly distributed repeat sequences, ingenomic nucleic acid.

2. Background

Nucleic acid hybridization is a fundamental process in molecularbiology. Probe-based assays are useful in the detection, identification,quantitation and/or analysis of nucleic acids. Nucleic acid probes havelong been used to analyze samples for the presence of nucleic acid frombacteria, fungi, virus or other organisms and are also useful inexamining genetically-based disease states or clinical conditions ofinterest. Nonetheless, nucleic acid probe-based assays have been slow toachieve commercial success. This lack of commercial success is, at leastpartially, the result of difficulties associated with specificity,sensitivity and/or reliability.

Fluorescence in-situ hybridization (FISH) has become an important toolfor determining the number, size and/or location of specific DNAsequences in mammalian cells. Typically, the hybridization reactionfluorescently stains the target sequences so that their location, sizeand/or number can be determined using fluorescence microscopy, flowcytometry or other suitable instrumentation. DNA sequences ranging fromwhole genomes down to several kilobases can be studied using currenthybridization techniques in combination with commercially availableinstrumentation.

In Comparative Genomic Hybridization (CGH) whole genomes are stained andcompared to normal reference genomes for the detection of regions withaberrant copy number. In the m-FISH technique (multi color FISH) eachseparate normal chromosome is stained by a separate color (Eils et al,Cytogenetics Cell Genet 82: 160-71 (1998)). When used on abnormalmaterial, the probes will stain the aberrant chromosomes therebydeducing the normal chromosomes from which they are derived (Macville Met al., Histochem Cell Biol. 108: 299-305 (1997)). Specific DNAsequences, such as the ABL gene, can be reliably stained using probes ofonly 15 kb (Tkachuk et al., Science 250: 559-62 (1990)). FISH-basedstaining is sufficiently distinct such that the hybridization signalscan be seen both in metaphase spreads and in interphase nuclei. Singleand multicolor FISH, using nucleic acid probes, have been applied todifferent clinical applications generally known as molecularcytogenetics, including prenatal diagnosis, leukemia diagnosis, andtumor cytogenetics.

A large component of the human genome comprises repeat sequences. Heatdenaturation and reannealing studies on DNA of higher organisms havedistinguished three populations of eukaryotic DNA; a quickly reannealingcomponent representing 25% of total DNA, an intermediate component thatrepresents 30% of the total DNA, and a slow component that represents45% of the total DNA (Britten et al., Science 161: 529-540 (1968)).Sequence analysis has shown that the slow component is made up bysingle-copy sequences, which include protein encoding genes, while thefast and intermediate components represents repetitive sequences. Thefast component contains small (a few nucleotides long), highlyrepetitive DNA sequences, which are usually found in tandem while theintermediate component contain the interspersed repetitive DNA (Novicket al., Human Genome Bioscience, 46(1): 32-41 (1996) and Brosius J.,Science 251: 753 (1991)). The repetitive units of the intermediatecomponent are interspersed within the genome and is the major reasonthat large genomic nucleic acid probes (i.e. >100 bp) derived fromgenomic nucleic acid are not well suited for hybridization analysis.

Interspersed repeated sequences are classified as either SINEs (shortinterspersed repeated sequences) or LINEs (Kroenberg et al., Cell, 53:391-400 (1988)). In primates, each of these classes are dominated by asingle DNA sequence family, both of which are classified as retrosponos(Rogers J., International Review of Cytology, 93: 187-279 (1985)). Themajor human SINEs are the Alu-repeat DNA sequence family. The Alu-repeatDNA family members are characterized by a consensus sequence ofapproximately 280 to 300 bp which consist of two similar sequencesarranged as a head to tail dimer. Approximately one million copies ofthe Alu repeat sequence are estimated to be present per haploid humangenome, thereby representing about ten percent of the genome (Ausubel etal., Current Protocols In Molecular Biology, John Wiley & Sons, Inc.,1996)). That estimate is consistent with the recent sequencedetermination of the human chromosome 21 and 22. These reportsdemonstrate that Alu repeats cover 9.48% and 16.80% of the DNA,respectively (Hattori et al. Nature, 405: 311-319 (2000) and Dunham I.et al., Nature, 402: 489-495 (1999)).

Alu elements have amplified in the human genome through retropositionover the past 65 million years and have been organized into a wealth ofoverlapping subfamilies based on diagnostic mutations shared bysubfamily members (See For Example: Batzer et al., J. Mol. Evol., 42:3-6 (1996)). Batzer et al. described a consensus nomenclature for Alurepeats sequences; representing the oldest (J), intermediate (S) andyoung (Y) family branches. Only the Y family branch is stilltranscriptional active but it is very small as each of the defined a5,a8 and b8 subfamily members have produced less than 2000 elements(Sherry et al., Genetics, 147: 1977-1982 (1997)). It has been calculatedthat of the primate Alu repeat family branches, approximately one-fifthbelong to the J family and four-fifths to the S family (Britten, R. J.,Proc. Natl. Acad. Sci. USA, 91: 6148-6150 (1994). The S family isdominated by the Sx subfamily as it represents more than 50% of thetotal S family branch.

In addition to SINEs and LINEs, there are several other types of repeatsthat are known to exist in genomic nucleic acid of humans as well as inother organisms. Chromosome telomeres are repeat sequences that appearto exist only, or else predominately, at the termini of all chromosomes.They are believed to shorten during the life of an organism and may playa role in the aging of an organism (See: Landsorp, P., WIPO PatentApplication No. WO97/14026). Likewise, chromosome centromeres containdistinct repeat sequences that exist only, or else predominately, in thecentral (centromere) region of a chromosome. Certain of the centromererepeat sequences can be detected in all chromosomes of an organismwhilst other repeat sequences are unique to a particular chromosome andcan be used to identify specific chromosomes (Taneja et al., Genes,Chromosomes & Cancer, 30: 57-63 (2001)).

Telomere and centromere repeat sequences differ from interspersedrepetitive sequence, such as SINE and LINEs, in that the telomere andcentromere repeat sequences are localized within a certain region of thechromosome. By comparison, SINEs and LINEs, which are referred to hereinas randomly distributed repeat sequences, are dispersed randomlythroughout the entire genome (Ullu E., TIBS: 216-219 (June, 1982)).Thus, as used herein, the term “randomly distribute repeat sequence” isintended to refer to repeat sequences that occur randomly within all, oressentially all, genomic nucleic acid of an organism. These include, butare not limited to, Alu-repeats, Kpn-repeats, di-nucleotide repeats,tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotiderepeats, hexa-nucleotide repeats, all of which are more generallyclassified as SINEs or LINEs.

Detection of specific nucleic acid sequences by in situ hybridizationusing non-radioactive labels has been applied for almost twenty years.As stated above however, the randomly distributed repeat sequences, suchas SINEs and LINEs, are particularly problematic for the production ofspecific nucleic acid probes that are derived from large clones becausethe probes will inevitably comprise randomly distributed repeatsequence. The problem arises because the nucleic acid probes will havethe randomly distributed repeat sequence contained therein, therebyfacilitating hybridization between the randomly distributed repeatsequences of the probes and natural genomic nucleic acid found withinall chromosomes. Because the detectable probes hybridize specifically tothe target, as well as to repeat sequence that is randomly found in thegenomic nucleic acid, there is a high degree of background signal thatis produced.

Refinement of non-radioactive detection and visualization methodsresulted in improved detection limits and thereby allowed thelocalization of large single-copy sequences (Landegent et al., Nature,317: 175-177 (1985)). In this study it was necessary to construct amixture of seven subclones (a total of 22.3 kb derived from a cosmid DNAclone containing the 3′ end of the Tg gene) in order to eliminate highlyrepeated sequences present in the original genomic cosmid DNA. Althoughthis was an improvement, a more attractive strategy, based on direct useof large genomic cloned segments in combination with Cot1 DNA, has beendescribed. The use of Cot1 DNA eliminates background signal, caused byhighly repetitive sequences, by introducing a competitive hybridizationprocess (Landegent et al., Hum. Genet., 77: 366-370 (1987); U.S. Pat.No. 5,447,841, issued to Gray et al.; and U.S. Pat. No. 6,203,977 B1issued to Ward et al.).

Cot1 DNA is a heterogeneous mixture of genomic nucleic acid that isprepared by degrading total human DNA and processing the resultingmaterial to thereby select for genomic nucleic acid fragments that areenriched in the repeat sequences (Britten et el., Methods Enzymol 29:363-418 (1986)). Although the use of Cot1 DNA has been proven to beeffective in suppressing undesired binding of detectable nucleic acidfragments of greater that 100 bp to target genomic nucleic acid, thereare several disadvantages to this method. One such disadvantage pertainsto the preparation of the Cot1 DNA itself. Specifically, because theprocess relies on the availability of total human DNA, the startingmaterial is itself not highly defined and is likely to vary from sampleto sample. Moreover, the processing methods are likely to producematerial that varies from batch to batch; this result being somewhatdependent upon the variability of the starting material and somewhatdependent upon the variability of the production process itself.Additionally, the Cot1 DNA is a heterogeneous mixture of fragments thatis impossible to completely characterize and define. Hence, the batch tobatch variability, as well as the inability to characterize the Cot1 DNAproduct, leaves substantial room for improvement. The present inventionaddresses these, as well as other, limitations of the art.

Despite its name, Peptide Nucleic Acid (PNA) is neither a peptide, anucleic acid nor is it an acid. Peptide Nucleic Acid (PNA) is anon-naturally occurring polyamide (pseudopeptide) that can hybridize tonucleic acid (DNA and RNA) with sequence specificity (See: U.S. Pat. No.5,539,082 and Egholm et al., Nature 365: 566-568 (1993)). Because theyhybridize to nucleic acid with sequence specificity, PNA oligomers havebecome commonly used in probe based applications for the analysis ofnucleic acids.

Being a non-naturally occurring molecule, unmodified PNA is not known tobe a substrate for the enzymes that are known to degrade peptides ornucleic acids. Therefore, PNA should be stable in biological samples, aswell as have a long shelf-life. Unlike nucleic acid hybridization, whichis very dependent on ionic strength, the hybridization of a PNA with anucleic acid is fairly independent of ionic strength and is favored atlow ionic strength, conditions that strongly disfavor the hybridizationof nucleic acid to nucleic acid (Egholm et al., Nature, at p. 567).Because of their unique properties, it is clear that PNA is not theequivalent of a nucleic acid in either structure or function.

Labeled PNA probes have been used for the analysis of rRNA in ISH andFISH assays (See: WO95/32305 and WO97/18325). Labeled PNA probes havealso been used in the analysis of mRNA (e.g. Kappa & Lambda Light Chain;Thisted M. et al., Cell Vision 3: 358-363 (1996)) and viral nucleic acid(e.g. EBV; Just T et al., J. Vir. Methods: 73: 163-174 (1998)). Alabeled PNA probe has also been used to detect human X chromosomespecific sequences in a PNA-FISH format (See: WO97/18325, now U.S. Pat.No. 5,888,733). The analysis of chromosome aberrations using PNA probeshas also been disclosed (See: WO99/57309). The ISH based analysis ofeukaryotic chromosomes and cells, using polyamide nucleic acids, hasalso been suggested (See: U.S. Pat. No. 5,888,734).

Labeled peptide nucleic acids have been described for the analysis ofboth telomere and centromere repeat sequences in genomic nucleic acid(Lansdorp, P., WO97/14026). Likewise, labeled peptide nucleic acidoligomers have been used in the analysis of individual human chromosomesin a multiplex PNA-FISH assay (Taneja et al., Genes, Chromosomes &Cancer, 30: 57-63 (2001). Similarly, the analysis of trinucleotiderepeats in chromosomal DNA, using appropriate labeled PNA probes, hasalso been suggested (See: WO97/14026). Subsequently, DNA and PNA probeswere used to examine cells for genetic defects associated with theexpansion of trinucleotide repeats that manifest as the disease known ashuman myotonic dystrophy (See: Taneja, Biotechniques, 24: 472-476(1998)). In all cases, labeled PNA probes were used to detect thespecific target nucleic acid repeat sequences.

PNA oligomers comprising the triplet repeat sequence CAG have also beenused for the selective isolation of transcriptionally active chromatinrestriction fragments (See: Boffa et al., Proc. Nat'l. Acad. Sci. USA,92: 1901-1905 (1995)).

Peptide nucleic acid oligomers have also been used to suppress thebinding of detectable probes to non-target sequences (See: U.S. Pat. No.6,110,676). Importantly however, there is no specific description,suggestion or teaching of using peptide nucleic acid oligomers tosuppress the binding of detectable nucleic acid probes to undesiredrandomly distributed repeat sequences of genomic nucleic acid.

SUMMARY OF THE INVENTION

Generally, this invention is directed to methods, kits, non-nucleotideprobes as well as other compositions pertaining to the suppression ofbinding of detectable nucleic acid probes to undesired nucleotidesequences of genomic nucleic acid in assays designed to determine targetgenomic nucleic acid. In many cases, the most problematic undesirednucleotide sequences are SINEs and LINEs (i.e. randomly distributedrepeat sequence), which include, but are not limited to, Alu-repeats,Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,tetra-nucleotide repeats, penta-nucleotide repeats and hexa-nucleotiderepeats.

In one embodiment, this invention pertains to a non-nucleotide probe ofat least sixteen nucleobase containing subunits in length having anaggregate nucleobase sequence that is at least eighty percent homologousto a sixteen nucleotide segment of randomly distributed repeat sequenceof genomic nucleic acid. By homologous, we mean nucleobase sequencehomology. By aggregate nucleobase sequence we mean the nucleobasesequence comprising the aggregate of nucleobase containing subunits ofthe probe even if separated by one or more linkers. The nucleobasesequence of the non-nucleotide probe can be substantially or completelyhomologous to a fraction, or part, of either: (i) a known unit repeat ofAlu-repeat sequence; or (ii) a consensus sequence of a known unit repeatof Alu-repeat sequence. The nucleobase sequence of the non-nucleotideprobe can be at least eighty percent homologous to a sixteen nucleotidesegment of the consensus unit repeat of Alu-repeat selected from thegroup consisting of: Seq. Id. No. 1 and Seq. Id. No. 2 (See: Table 1).The non-nucleotide probe can be a peptide nucleic acid.

In another embodiment, this invention pertains to a non-nucleotide probecontaining an aggregate nucleobase sequence of at least ten consecutivenucleobases that is at least eighty percent homologous to the nucleobasesequences selected from the group consisting of: Seq. Id. No. 3, Seq.Id. No. 4, Seq. Id. No. 5, Seq. Id. No. 6, Seq. Id. No. 7, Seq. Id. No.8, Seq. Id. No. 9, Seq. Id. No. 10, Seq. Id. No. 11, Seq. Id. No. 12,Seq. Id. No. 13, Seq. Id. No. 14, Seq. Id. No. 15, Seq. Id. No. 16, Seq.Id. No. 17, Seq. Id. No. 18, Seq. Id. No. 19, Seq. Id. No. 20, Seq. Id.No. 21, Seq. Id. No. 22, Seq. Id. No. 23, Seq. Id. No. 24, Seq. Id. No.25 and Seq. Id. No. 26 (See: Table 1). These particular sequences, ortheir complements, have been determined to be highly effective atsuppressing the binding of a detectable nucleic acid probe to undesiredchromosomes or chromosome regions in an assay for detecting the ERBB2(alias HER2) or MLL target nucleic acid sequence in genomic nucleic acid(See: Examples 4 and 5). The ten consecutive nucleobases can be either:(i) at least ninety percent homologous to the identified sequences; or(ii) exactly homologous to the identified sequences. The probe can beidentical in nucleobase sequence to any one of the identified sequences.The non-nucleotide probe can be a peptide nucleic acid oligomer.

In still another embodiment, this invention pertains to a mixture of twoor more non-nucleotide probes wherein each probe contains an aggregatenucleobase sequence that is at least eighty percent homologous to asixteen nucleotide segment of randomly distributed repeat sequence ofgenomic nucleic acid. The randomly distributed repeat sequence can be aSINE or LINE. SINEs and LINEs can be selected from the group consistingof: Alu-repeats, Kpn-repeats, di-nucleotide repeats, tri-nucleotiderepeats, tetra-nucleotide repeats, penta-nucleotide repeats andhexa-nucleotide repeats. The non-nucleotide probes can be peptidenucleic acid oligomers. The mixture of probes can further comprise oneor more detectable nucleic acid probes.

In yet another embodiment, this invention pertains to a compositioncomprising genomic nucleic acid containing one or more segments ofrandomly distributed repeat sequence. The randomly distributed repeatsequence can be a SINE or LINE. SINEs and LINES can be selected from thegroup consisting of: Alu-repeats, Kpn-repeats, di-nucleotide repeats,tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotiderepeats and hexa-nucleotide repeats. The composition can furthercomprise two or more different non-nucleotide probes that sequencespecifically hybridized to at least a fraction, or part, of the one ormore segments of randomly distributed repeat sequence of the genomicnucleic acid. Hence, the composition can be the hybrid of the segment ofrandomly distributed repeat sequence and the two or more non-nucleotideprobes. The non-nucleotide probes can be peptide nucleic acid oligomers.

Because it may be desirable to provide the mixture of non-nucleotideprobes in the same container as the detectable nucleic acid probes, andbecause the detectable nucleic acid probes can possess segments ofnucleobase sequence that are derived from the randomly distributedrepeat sequences, this invention is further directed to a compositioncomprising a detectable nucleic acid probe of at least 100 bp that hasbeen derived from genomic nucleic acid and that contains one or moresegments of randomly distributed repeat sequence. The randomlydistributed repeat sequences can be SINEs or LINEs. The SINEs or LINEscan be selected from the group consisting of: Alu-repeats, Kpn-repeats,di-nucleotide repeats, tri-nucleotide repeats, tetra-nucleotide repeats,penta-nucleotide repeats and hexa-nucleotide repeats. The compositioncan further comprise two or more different non-nucleotide probessequence specifically hybridized to at least a fraction of the one ormore segments of randomly distributed repeat sequence of the detectablenucleic acid probe. The non-nucleotide probes can be peptide nucleicacid oligomers.

In still another embodiment, this invention is directed to a method forsuppressing the binding of one or more detectable nucleic acid probes,that are greater than 100 bp and that have been derived from genomicnucleic acid, to one or more undesired sequences in an assay fordetermining target genomic nucleic acid of a sample. The methodcomprises contacting the sample with a mixture of two or morenon-nucleotide probes wherein each probe contains an aggregatenucleobase sequence that is at least eighty percent homologous to asegment of randomly distributed repeat sequence of genomic nucleic acid.According to the method, the sample is also contacted with the one ormore detectable nucleic acid probes. The target genomic nucleic acid ofthe sample can then determined by determining the hybridization of theone or more detectable nucleic acid probes to the target genomic nucleicacid of the sample wherein the presence, absence or amount ofhybridization of the detectable nucleic acid probe to the target genomicnucleic acid is representative of the presence, absence or amount oftarget genomic nucleic acid in the sample. The non-nucleotide probes canbe peptide nucleic acid oligomers.

Thus, in yet another embodiment, this invention pertains to comparing asample of genomic nucleic acid with that of a control sample using agenomic nucleic acid reference array. The method comprises providing asample of genomic nucleic acid to be tested, providing a control ofgenomic nucleic acid, wherein the control and the sample aredifferentially labeled, providing a genomic nucleic acid referencearray, and providing a mixture of two or more non-nucleotide probeswherein each probe contains an aggregate nucleobase sequence that is atleast eighty percent homologous to a sixteen nucleotide segment ofrandomly distributed repeat sequence of genomic nucleic acid. The methodfurther comprises treating the sample and control genomic nucleic acid,the array or both the sample and control genomic nucleic acid and thearray with the mixture of non-nucleotide probes under suitablehybridization conditions. The array can then be contacted with thetreated mixture of sample and control genomic nucleic acid undersuitable hybridization conditions. The intensities of the signals fromthe differential labels on the array, caused by hybridization of theprobes to genomic nucleic acid, can then be compared to therebydetermine one or more variations in copy numbers of sequences in thesample as compared with the relative copy numbers of substantiallyidentical sequences in the control.

In still another embodiment, this invention is directed to a method fordetermining non-nucleotide probes that hybridize to randomly distributedrepeat sequences and that are suitable for suppressing the binding of adetectable nucleic acid probe, that is greater than 100 bp in length andthat is derived from genomic nucleic acid, to one or more undesirablesequences in an assay for determining target genomic nucleic acid of asample. The method comprises designing possible nucleobase sequences ofnon-nucleotide probes using sequence alignment of known randomlydistributed repeat sequences and then preparing labeled non-nucleotideprobes having said possible nucleobase sequences. According to themethod, genomic nucleic acid of a sample that contains the targetgenomic nucleic acid can be treated with the labeled non-nucleotideprobes under suitable hybridization conditions. The relative signal ofthe hybridized labeled probes of the many different possible nucleobasesequences can then be determined. Based upon the signal intensity data,the probe or probes that exhibit the strongest signal, as a result ofbinding to the genomic nucleic acid, can be selected and tested tothereby determine whether or not they are suitable for suppressing thebinding of a detectable nucleic acid probe of greater than 100 bp inlength that is derived from genomic nucleic acid to one or morenon-target sequences in an assay for determining target genomic nucleicacid of a sample. In order to test the probe or probes, they can bere-synthesized in unlabeled form and then tested using the method forsuppressing the binding of detectable probes to undesired sequences asdescribed above. The non-nucleotide probes can be peptide nucleic acidoligomers.

In still another embodiment, this invention is directed to a reagent kitcomprising a mixture of two or more non-nucleotide probes containing atleast sixteen consecutive nucleobases that are at least eighty percenthomologous to a fraction of the unit repeat Alu-repeat consensussequence selected from the group consisting of: Seq. Id. No. 1 or Seq.Id. No. 2. The kit further comprises other reagents, compositions and/orinstructions suitable for performing an assay to thereby determinegenomic nucleic acid of a sample. The reagent kit can further compriseone or more detectable nucleic acid probes of greater than 100 bp inlength and that are derived from genomic nucleic acid. The one or moredetectable nucleic acid probes can be provided in the container thatcontains the mixture of two or more non-nucleotide probes.

In yet still another embodiment, this invention is directed to a kitcomprising a mixture of two or more non-nucleotide probes wherein atleast one probe contains a segment of at least ten consecutivenucleobases that are at least eighty percent homologous to theAlu-repeat sequences selected from the group consisting of: Seq. Id. No.3, Seq. Id. No. 4, Seq. Id. No. 5, Seq. Id. No. 6, Seq. Id. No. 7, Seq.Id. No. 8, Seq. Id. No. 9, Seq. Id. No. 10, Seq. Id. No. 11, Seq. Id.No. 12, Seq. Id. No. 13, Seq. Id. No. 14, Seq. Id. No. 15, Seq. Id. No.16, Seq. Id. No. 17, Seq. Id. No. 18, Seq. Id. No. 19, Seq. Id. No. 20,Seq. Id. No. 21, Seq. Id. No. 22, Seq. Id. No. 23, Seq. Id. No. 24, Seq.Id. No. 25 and Seq. Id. No. 26 (See: Table 1). The kit further comprisesother reagents, compositions and/or instructions for performing a assayto thereby determine genomic nucleic acid of a sample. The kit canfurther comprise one or more detectable nucleic acid probes of greaterthan 100 bp in length and that are derived from genomic nucleic acid. Ina most preferred embodiment, one or more detectable nucleic acid probescan be provided in the container that contains the mixture of two ormore non-nucleotide probes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the positioning of probes on the individualstrands within complementary strands of a unit repeat of an Alu-repeatconsensus sequence.

FIGS. 2-1, 2-2 and 2-3 are together output from sequence alignmentsoftware with the five Alu sequences HSU14574.seq Sx (Seq. Id. No. 30),HSU14573.seq Sq (Seq. Id. No. 31), HSU14572.seq Sp (Seq. Id. No. 32),HSU14571.seq Sc (Seq. Id. No. 33), and HSU14567.seq J (Seq. Id. No. 34))and the Majority consensus sequence (SEQ ID NO. 29).

FIGS. 3A-1, 3A-2, 3B-1 and 3B-2 are microscope generated images ofinterphase nuclei and metaphase spread of human chromosomes treated withlabeled PNA oligomers that either obtained a high R-banding score (3A-1& 3A-2) or that just passed the defined lower limit for a suitableR-banding pattern score (3B-1 & 3B-2).

FIGS. 4A, 4B, 4C and 4D are microscope generated images of a metaphasespread of human chromosomes treated with labeled PNA oligomers designedfrom the J and S family consensus (4A & 4B) or the Y family consensus(4C & 4D).

FIGS. 5A, 5B and 5C are microscope generated images of interphase nucleiand metaphase spread of human chromosomes treated with detectable HER-2nucleic acid probe and either: 1) a blocking mixture of unlabeled PNAoligomers (5A); 2) Cot1 DNA blocker (5B) or is otherwise not treatedwith a blocking reagent (5C).

FIGS. 6A, 6B and 6C are microscope generated images of interphase nucleiand metaphase spread of human chromosomes treated with detectable MLLnucleic acid probe and either: 1) a blocking mixture of unlabeled PNAoligomers (6A); 2) Cot1 DNA blocker (6B) or is otherwise not treatedwith a blocking reagent (6C).

FIGS. 7A, 7B and 7C are microscope generated images of paraffin embeddedtissue sections of a breast carcinoma treated with detectable HER2nucleic acid probe and either: 1) a blocking mixture of unlabeled PNAoligomers (7A); 2) Cot-1 DNA blocker (7B) or is otherwise not treatedwith a blocking reagent (7C).

FIGS. 8A, 8B, 8C and 8D are microscope generated images of interphasenuclei and metaphase spreads treated with detectable HER2 nucleic acidprobe and either: 1) no other probes (8A); 2) a mixture of Alu PNABlocking Probes (8B); Chromosome 17 PNA Probes 8C); or Alu PNA BlockingProbes and Chromosome 17 PNA Probes (8D).

FIGS. 9A, 9B, 9C, 9D, 9E and 9F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine theCyclinD1 gene.

FIGS. 10A, 10B, 10C, 10D, 10E and 10F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine the c-MYCgene.

FIGS. 11A, 11B, 11C, 11D, 11E and 11F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine the EGFRgene.

FIGS. 12A, 12B, 12C, 12D, 12E, 12F, 12G, 12H and 12I are microscopegenerated images of interphase nuclei and metaphase spreads treated todetermine the TOP2A deletion.

FIGS. 13A, 13B, 13C, 13D, 13E and 13F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine the TELgene.

FIGS. 14A, 14B, 14C, 14D, 14E and 14F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine the E2Agene.

FIGS. 15A, 15B, 15C, 15D, 15E and 15F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine the BCRgene.

FIGS. 16A, 16B, 16C, 16D, 16E and 16F are microscope generated images ofinterphase nuclei and metaphase spreads treated to determine the IGHgene.

FIGS. 17A, 17B and 17C are microscope generated images of interphasenuclei and metaphase spreads treated to determine the IGL gene.

FIGS. 18A, 18B and 18C are microscope generated images of interphasenuclei and metaphase spreads treated to determine the IGK gene.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

For the purposes of interpreting this specification the followingdefinitions shall apply and whenever appropriate, terms used in thesingular shall also include the plural and vice versa.

a. As used herein, “nucleobase” means those naturally occurring andthose non-naturally occurring heterocyclic moieties commonly known tothose who utilize nucleic acid technology or utilize peptide nucleicacid technology to thereby generate polymers that can sequencespecifically bind to nucleic acids. Non-limiting examples of suitablenucleobases include: adenine, cytosine, guanine, thymine, uracil,5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). Other non-limiting examples of suitablenucleobases include those nucleobases illustrated in FIGS. 2(A) and 2(B)of Buchardt et al. (U.S. Pat. No. 6,357,163).b. As used herein, “sequence specifically” means hybridization by basepairing through hydrogen bonding. Non-limiting examples of standard basepairing includes adenine base pairing with thymine or uracil and guaninebase pairing with cytosine. Other non-limiting examples of base-pairingmotifs include, but are not limited to: adenine base pairing with anyof: 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 2-thiouracil or2-thiothymine; guanine base pairing with any of: 5-methylcytosine orpseudoisocytosine; cytosine base pairing with any of: hypoxanthine,N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine); thymine or uracilbase pairing with any of: 2-aminopurine, N9-(2-amino-6-chloropurine) orN9-(2,6-diaminopurine); and N8-(7-deaza-8-aza-adenine), being auniversal base, base pairing with any other nucleobase, such as forexample any of: adenine, cytosine, guanine, thymine, uracil,5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine) or N9-(7-deaza-8-aza-guanine) (See: Seela et al.,Nucl. Acids, Res.: 28(17): 3224-3232 (2000)).c. As used herein, “nucleobase sequence” means all or a segment ofnucleobase-containing subunits in an oligomer or polymer. Non-limitingexamples of suitable polymers or polymers segments that comprise“nucleobase sequence” or “nucleobase sequences” includeoligodeoxynucleotides (e.g. DNA), oligoribonucleotides (e.g. RNA),peptide nucleic acids (PNA), nucleic acid analogs and/or nucleic acidmimics.d. As used herein, “target sequence” or “target genomic nucleic acid” isa nucleobase sequence sought to be determined. The nucleobase sequencecan be a subsequence of a nucleic acid molecule of interest (e.g. achromosome).e. As used herein, “nucleic acid” is a nucleobase sequence-containingoligomer, polymer, or polymer segment, having a backbone formed solelyfrom nucleotides, or analogs thereof. Preferred nucleic acids are DNAand RNA. For the avoidance of doubt, a peptide nucleic acid (PNA)oligomer is not a nucleic acid since it is not formed from nucleotidesor analogs thereof.f. As used herein, “nucleotide” means any of several compounds thatconsist of a ribose or deoxyribose sugar joined to a purine orpyrimidine base and to a phosphate group. Nucleotides are the basicstructural subunits of nucleic acids (e.g. RNA and DNA).g. As used herein, “analog” or “nucleic acid analog” means an oligomer,polymer, or polymer segment composed of at least one modifiednucleotide, or subunits derived directly a modification of nucleotides.h. As used herein, “mimic” of “nucleic acid mimic” means anon-nucleotide polymer.i. As used herein, a “non-nucleotide polymer” or “non-nucleotide probe”is a nucleobase sequence-containing oligomer, polymer, or polymersegment that does not comprise nucleotides. A most preferrednon-nucleotide polymer is a peptide nucleic acid (PNA) oligomer.j. As used herein, “peptide nucleic acid” or “PNA” means any oligomer orpolymer comprising two or more PNA subunits (residues), including, butnot limited to, any of the oligomer or polymer segments referred to orclaimed as peptide nucleic acids in U.S. Pat. Nos. 5,539,082, 5,527,675,5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855,5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, 6,107,4706,201,103, 6,228,982 and 6,357,163; all of which are herein incorporatedby reference. The term “peptide nucleic acid” or “PNA” shall also applyto any oligomer or polymer segment comprising two or more subunits ofthose nucleic acid mimics described in the following publications:Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4:1081-1082 (1994); Petersen et al., Bioorganic & Medicinal ChemistryLetters, 6: 793-796 (1996); Diderichsen et al., Tett. Lett. 37: 475-478(1996); Fujii et al., Bioorg. Med. Chem. Lett. 7: 637-627 (1997); Jordanet al., Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz et al., Tett.Lett. 36: 6941-6944 (1995); Lagriffoul et al., Bioorg. Med. Chem. Lett.4: 1081-1082 (1994); Diederichsen, U., Bioorganic & Medicinal ChemistryLetters, 7: 1743-1746 (1997); Lowe et al., J. Chem. Soc. Perkin Trans.1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11:547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1 1:5 55-560(1997); Howarth et al., J. Org. Chem. 62: 5441-5450 (1997); Altmann, K-Het al., Bioorganic & Medicinal Chemistry Letters, 7: 1119-1122 (1997);Diederichsen, U., Bioorganic & Med. Chem. Lett., 8: 165-168 (1998);Diederichsen et al., Angew. Chem. Int. Ed., 37: 302-305 (1998); Cantinet al., Tett. Lett., 38: 4211-4214 (1997); Ciapetti et al., Tetrahedron,53: 1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3: 912-919(1997); Kumar et al., Organic Letters 3(9): 1269-1272 (2001); and thePeptide-Based Nucleic Acid Mimics (PENAMs) of Shah et al. as disclosedin WO96/04000.

In certain embodiments, a “peptide nucleic acid” or “PNA” is an oligomeror polymer segment comprising two or more covalently linked subunits ofthe formula:

wherein, each J is the same or different and is selected from the groupconsisting of H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I. Each K isthe same or different and is selected from the group consisting of O, S,NH and NR¹. Each R¹ is the same or different and is an alkyl grouphaving one to five carbon atoms that may optionally contain a heteroatomor a substituted or unsubstituted aryl group. Each A is selected fromthe group consisting of a single bond, a group of the formula;—(CJ₂)_(s)- and a group of the formula; —(CJ₂)_(s)C(O)—, wherein, J isdefined above and each s is a whole number from one to five. Each t is 1or 2 and each u is 1 or 2. Each L is the same or different and isindependently selected from: adenine, cytosine, guanine, thymine,uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine), other naturally occurring nucleobase analogsor other non-naturally occurring nucleobases.

In certain other embodiments, a PNA subunit consists of a naturallyoccurring or non-naturally occurring nucleobase attached to theN-α-glycine nitrogen of the N-[2-(aminoethyl)]glycine backbone through amethylene carbonyl linkage; this currently being the most commonly usedform of a peptide nucleic acid subunit.

k. As used herein, the terms “label”, “reporter moiety” or “detectablemoiety” are interchangeable and refer to moieties that can be attachedto an oligomer or polymer to thereby render the oligomer or polymerdetectable by an instrument or method.

l. As used herein, “stained” means that individual organisms,chromosomes or chromosome fragments or segments are directly orindirectly marked with a detectable moiety as a result of the sequencespecific hybridization thereto of one or more detectable probes.m. As used herein, “unit repeat” means the basic unit of nucleobasesequence that is repeated in a randomly distributed repeat sequence.n. As used herein, “block”, “oligomer block” or “block oligomer” areinterchangeable and all mean a PNA oligomer that is designed andavailable to be ligated to a second appropriately modified PNA oligomerto thereby prepare an elongated PNA oligomer. Oligomers or blocks thatare ligated/condensed may be unlabeled, labeled with one or morereporter moieties and/or comprise one or more protected or unprotectedfunctional groups. With respect to an elongated oligomer, “block” canalso be used to refer to a part of the elongated oligomer thatoriginates from an oligomer block used to form the elongated oligomer.The elongated oligomer also may be used as a block in aligation/condensation reaction that further elongates the PNA oligomer.

2. Description of the Invention I. General

Production, Purification & Labeling of Detectable Nucleic Acid Probes

To amplify a specific DNA sequence by cloning, the DNA can be insertedinto a vector and both insert and vector can then be amplified insideappropriate host cells. The amplified DNA can then extracted. Commonlyused vectors include bacterial plasmids, cosmids, PACs, BACs, and YACs.

The purified DNA can be labeled with different methods, e.g. enzymaticor chemical. The most frequently used method is Nick translation. TheNick translation reaction can employ two enzymes, Dnase I which producesthe “nicks” in the double-stranded DNA and DNA polymerase, whichincorporates labeled nucleotides along both strands of the DNA duplex.Any labeling method known to those in the art can be used for labelingthe nucleic acid probe as used in this invention.

PNA Synthesis:

Methods for the chemical assembly of PNAs are well known (See: U.S. Pat.Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336,5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610,5,986,053, 6,107,470, 6,201,103, 6,228,982 and 6,357,163; all of whichare herein incorporated by reference (Also see: PerSeptive BiosystemsProduct Literature)). As a general reference for PNA synthesismethodology also please see: Nielsen et al., Peptide Nucleic Acids;Protocols and Applications, Horizon Scientific Press, Norfolk England(1999).

Chemicals and instrumentation for the support bound automated chemicalassembly of peptide nucleic acids are now commercially available. Bothlabeled and unlabeled PNA oligomers are likewise available fromcommercial vendors of custom PNA oligomers. Chemical assembly of a PNAis analogous to solid phase peptide synthesis, wherein at each cycle ofassembly the oligomer possesses a reactive alkyl amino terminus that iscondensed with the next synthon to be added to the growing polymer.

PNA may be synthesized at any scale, from submicromole to millimole, ormore. PNA can be conveniently synthesized at the 2 μmole scale, usingFmoc (Bhoc), tBoc/Z, or MmT protecting group monomers on an ExpediteSynthesizer (Applied Biosystems) using a XAL or PAL support.Alternatively the Model 433A Synthesizer (Applied Biosystems) with MBHAsupport can be used. Moreover, many other automated synthesizers andsynthesis supports can be utilized. Because standard peptide chemistryis utilized, natural and non-natural amino acids can be routinelyincorporated into a PNA oligomer. Because a PNA is a polyamide, it has aC-terminus (carboxyl terminus) and an N-terminus (amino terminus). Forthe purposes of the design of a hybridization probe suitable forantiparallel binding to the target sequence (the preferred orientation),the N-terminus of the probing nucleobase sequence of the PNA probe isthe equivalent of the 5′-hydroxyl terminus of an equivalent DNA or RNAoligonucleotide.

PNA oligomers can also be prepared by the ligation of shorter oligomers,with (See: WO02/072865) or without (See: U.S. Ser. No. 60/409/220) theintroduction of a linker contained therebetween the oligomer blocks.

PNA Labeling/Modification:

Non-limiting methods for labeling PNAs are described in U.S. Pat. No.6,110,676, U.S. Pat. No. 6,280,964, U.S. Pat. No. 6,355,421, WO99/21881,U.S. Pat. No. 6,361,942, WO99/49293 and U.S. Pat. No. 6,441,152 (all ofwhich are herein incorporated by reference), the examples section ofthis specification or are otherwise well known in the art of PNAsynthesis and peptide synthesis. Methods for labeling PNA are alsodiscussed in Nielsen et al., Peptide Nucleic Acids; Protocols andApplications, Horizon Scientific Press, Norfolk, England (1999).Non-limiting methods for labeling PNA oligomers are discussed below.

Because the synthetic chemistry of assembly is essentially the same, anymethod commonly used to label a peptide can often be adapted to effectthe labeling a PNA oligomer. Generally, the N-terminus of the oligomeror polymer can be labeled by reaction with a moiety having a carboxylicacid group or activated carboxylic acid group. One or more spacermoieties can optionally be introduced between the labeling moiety andthe nucleobase containing subunits of the oligomer. Generally, thespacer moiety can be incorporated prior to performing the labelingreaction. If desired, the spacer may be embedded within the label andthereby be incorporated during the labeling reaction.

Typically the C-terminal end of the polymer can be labeled by firstcondensing a labeled moiety or functional group moiety with the supportupon which the PNA oligomer is to be assembled. Next, the firstnucleobase containing synthon of the PNA oligomer can be condensed withthe labeled moiety or functional group moiety. Alternatively, one ormore spacer moieties (e.g. 8-amino-3,6-dioxaoctanoic acid; the“O-linker”) can be introduced between the label moiety or functionalgroup moiety and the first nucleobase subunit of the oligomer. Once themolecule to be prepared is completely assembled, labeled and/ormodified, it can be cleaved from the support deprotected and purifiedusing standard methodologies.

For example, the labeled moiety or functional group moiety can be alysine derivative wherein the ϵ-amino group is a protected orunprotected functional group or is otherwise modified with a reportermoiety. The reporter moiety could be a fluorophore such as5(6)-carboxyfluorescein, Dye1, Dye2 or a quencher moiety such as4-((4-(dimethylamino)phenyl)azo)benzoic acid (dabcyl). Condensation ofthe lysine derivative with the solid support can be accomplished usingstandard condensation (peptide) chemistry. The α-amino group of thelysine derivative can then be deprotected and the nucleobase sequenceassembly initiated by condensation of the first PNA synthon with theα-amino group of the lysine amino acid. As discussed above, a spacermoiety may optionally be inserted between the lysine amino acid and thefirst PNA synthon by condensing a suitable spacer (e.g.Fmoc-8-amino-3,6-dioxaoctanoic acid) with the lysine amino acid prior tocondensation of the first PNA synthon.

Alternatively, a functional group on the assembled, or partiallyassembled, polymer can be introduced while the oligomer is still supportbound. The functional group will then be available for any purpose,including being used to either attached the oligomer to a support orotherwise be reacted with a reporter moiety, including being reactedpost-ligation (by post-ligation we mean at a point after the oligomerhas been fully formed by the performing of one or morecondensation/ligation reactions). This method, however, requires that anappropriately protected functional group be incorporated into theoligomer during assembly so that after assembly is completed, a reactivefunctional can be generated. Accordingly, the protected functional groupcan be attached to any position within the oligomer or block, including,at the oligomer termini, at a position internal to the oligomer.

For example, the ϵ-amino group of a lysine could be protected with a4-methyl-triphenylmethyl (Mtt), a 4-methoxy-triphenylmethyl (MMT) or a4,4′-dimethoxytriphenylmethyl (DMT) protecting group. The Mtt, MMT orDMT groups can be removed from the oligomer (assembled usingcommercially available Fmoc PNA monomers and polystyrene support havinga PAL linker; PerSeptive Biosystems, Inc., Framingham, Mass.) bytreatment of the synthesis resin under mildly acidic conditions.Consequently, a donor moiety, acceptor moiety or other reporter moiety,for example, can then be condensed with the ϵ-amino group of the lysineamino acid while the polymer is still support bound. After completeassembly and labeling, the polymer can then cleaved from the support,deprotected and purified using well-known methodologies.

By still another method, the reporter moiety can be attached to theoligomer or oligomer block after it is fully assembled and cleaved fromthe support. This method is preferable where the label is incompatiblewith the cleavage, deprotection or purification regimes commonly used tomanufacture the oligomer. By this method, the PNA oligomer can belabeled in solution by the reaction of a functional group on the polymerand a functional group on the label. Those of ordinary skill in the artwill recognize that the composition of the coupling solution will dependon the nature of oligomer and label, such as for example a donor oracceptor moiety. The solution may comprise organic solvent, water or anycombination thereof. Generally, the organic solvent will be a polarnon-nucleophilic solvent. Non limiting examples of suitable organicsolvents include acetonitrile (ACN), tetrahydrofuran, dioxane, methylsulfoxide, N,N′-dimethylformamide (DMF) and 1-methylpyrrolidone (NMP).

The functional group on the polymer to be labeled can be a nucleophile(e.g. an amino group) and the functional group on the label can be anelectrophile (e.g. a carboxylic acid or activated carboxylic acid). Itis however contemplated that this can be inverted such that thefunctional group on the polymer can be an electrophile (e.g. acarboxylic acid or activated carboxylic acid) and the functional groupon the label can be a nucleophile (e.g. an amino acid group).Non-limiting examples of activated carboxylic acid functional groupsinclude N-hydroxysuccinimidyl esters. In aqueous solutions, thecarboxylic acid group of either of the PNA or label (depending on thenature of the components chosen) can be activated with a water solublecarbodiimide. The reagent,1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC), is acommercially available reagent sold specifically for aqueous amideforming condensation reactions. Such condensation reactions can also beimproved when 1-Hydroxy-7-azabenzotriazole (HOAt) or1-hydroxybenzotriazole (HOBt) is mixed with the EDC.

The pH of aqueous solutions can be modulated with a buffer during thecondensation reaction. For example, the pH during the condensation canbe in the range of 4-10. Generally, the basicity of non-aqueousreactions will be modulated by the addition of non-nucleophilic organicbases. Non-limiting examples of suitable bases includeN-methylmorpholine, triethylamine and N,N-diisopropylethylamine.Alternatively, the pH can be modulated using biological buffers such as(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid) (HEPES) or4-morpholineethane-sulfonic acid (MES) or inorganic buffers such assodium bicarbonate.

Labeled Oligomers & Oligomer Blocks:

As discussed above, PNA oligomers can be labeled with reporter moieties.Non-limiting examples of reporter moieties (labels) suitable fordirectly labeling oligomers or oligomer blocks include: a quantum dot, aminor groove binder, a dextran conjugate, a branched nucleic aciddetection system, a chromophore, a fluorophore, a quencher, a spinlabel, a radioisotope, an enzyme, a hapten, an acridinium ester and achemiluminescent compound. Quenching moieties are also consideredlabels. Other suitable labeling reagents and preferred methods ofattachment would be recognized by those of ordinary skill in the art ofPNA, peptide or nucleic acid synthesis. Non-limiting examples aredescribed or referred to above.

Non-limiting examples of haptens include 5(6)-carboxyfluorescein,2,4-dinitrophenyl, digoxigenin, and biotin.

Non-limiting examples of fluorochromes (fluorophores) include5(6)-carboxyfluorescein (Flu), 2′,4′,1,4-tetrachlorofluorescein; and2′,4′,5′,7′,1,4-hexachlorofluorescein, other fluorescein dyes (See: U.S.Pat. Nos. 5,188,934; 6,008,379; 6,020,481, incorporated herein byreference), 6-((7-amino-4-methylcoumarin-3-acetyl)amino)hexanoic acid(Cou), 5(and 6)-carboxy-X-rhodamine (Rox), other rhodamine dyes (See:U.S. Pat. Nos. 5,366,860; 5,847,162; 5,936,087; 6,051,719; 6,191,278;6,248,884, incorporated herein by reference), benzophenoxazines (See:U.S. Pat. No. 6,140,500, incorporated herein by reference) Cyanine 2(Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5)Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye(Cyanine dyes 2, 3, 3.5, 5 and 5.5 are available as NHS esters fromAmersham, Arlington Heights, Ill.), other cyanine dyes (Kubista, WO97/45539), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE),5(6)-carboxy-tetramethyl rhodamine (Tamara), Dye 1 (FIG. 7), Dye2 (FIG.7) or the Alexa dye series (Molecular Probes, Eugene, Oreg.).

Non-limiting examples of enzymes include polymerases (e.g. Taqpolymerase, Klenow PNA polymerase, T7 DNA polymerase, Sequenase, DNApolymerase 1 and phi29 polymerase), alkaline phosphatase (AP),horseradish peroxidase (HRP), soy bean peroxidase (SBP)), ribonucleaseand protease.

Non-limiting examples of quenching moieties include diazo-containingmoieties such as aryldiazo compounds, e.g. dabcyl and dabsyl, homologscontaining one more additional diazo and/or aryl groups; e.g. FastBlack, (Nardone, U.S. Pat. No. 6,117,986), and substituted compoundswhere Z is a substituent such Cl, F, Br, C₁-C₆ alkyl, C₅-C₁₄ aryl,nitro, cyano, sulfonate, NR₂, —OR, and CO₂H, where each R isindependently H, C₁-C₆ alkyl or C₅-C₁₄ aryl according to the structures:

cyanine dyes (Lee, U.S. Pat. No. 6,080,868), including the exemplarystructure:

and other chromophores such as anthraquinone, malachite green,nitrothiazole, and nitroimidazole compounds and the like wherein thegroup X is the covalent attachment site of a bond or linker to theoligomers of the invention.

A non-limiting example of a minor groove binder is CDPI₃, represented bythe structure:

where X are exemplary attachment sites to a oligomer (Dempcy, WO01/31063).

Non-radioactive labeling methods, techniques, and reagents are reviewedin: Non-Radioactive Labeling, A Practical Introduction, Garman, A. J.Academic Press, San Diego, Calif. (1997).

Detectable and Independently Detectable Moieties/Multiplex Analysis:

In preferred embodiments of this invention, a multiplex hybridizationassay is performed. In a multiplex assay, numerous conditions ofinterest are simultaneously or sequentially examined. Multiplex analysisrelies on the ability to sort sample components or the data associatedtherewith, during or after the assay is completed. In one embodiments,one or more distinct independently detectable moieties can be used tolabel two or more different detectable nucleic acid probes used in anassay. The ability to differentiate between and/or quantitate each ofthe independently detectable moieties provides the means to multiplex ahybridization assay because the data correlates with the hybridizationof each of the distinct, independently labeled oligomer to a particulartarget sequence sought to be detected in the sample. Consequently, themultiplex assays of this invention may be used to simultaneously orsequentially detect the presence, absence, number, position and/oridentity of two or more target sequences in the same sample and/or inthe same assay.

Spacer/Linker Moieties:

Generally, spacers are used to minimize the adverse effects that bulkylabeling reagents might have on the hybridization properties of probesor primers. A linker is used to link two or more segments of an oligomeror polymer. Non-limiting examples of spacer/linker moieties used in thisinvention consist of: one or more aminoalkyl carboxylic acids (e.g.aminocaproic acid) the side chain of an amino acid (e.g. the side chainof lysine or ornithine) one or more natural amino acids (e.g. glycine),aminooxyalkylacids (e.g. 8-amino-3,6-dioxaoctanoic acid), alkyl diacids(e.g. succinic acid), alkyloxy diacids (e.g. diglycolic acid) oralkyldiamines (e.g. 1,8-diamino-3,6-dioxaoctane). Spacer/linker moietiesmay also incidentally or intentionally be constructed to improve thewater solubility of the oligomer (For example see: Gildea et al., Tett.Lett. 39: 7255-7258 (1998)).

Hybridization Conditions/Stringency:

Those of ordinary skill in the art of hybridization will recognize thatfactors commonly used to impose or control stringency of hybridizationinclude formamide concentration (or other chemical denaturant reagent),salt concentration (i.e., ionic strength), hybridization temperature,detergent concentration, pH and the presence or absence of chaotropes.Optimal stringency for a probe/target sequence combination is oftenfound by the well-known technique of fixing several of theaforementioned stringency factors and then determining the effect ofvarying a single stringency factor. The same stringency factors can bemodulated to thereby control the stringency of hybridization of a PNA toa nucleic acid, except that the hybridization of a PNA is fairlyindependent of ionic strength. Optimal stringency for an assay may beexperimentally determined by examination of each stringency factor untilthe desired degree of discrimination is achieved.

Suitable Hybridization Conditions:

Generally, the more closely related the background causing nucleic acidcontaminates are to the target sequence, the more carefully stringencymust be controlled. Suitable hybridization conditions will thus compriseconditions under which the desired degree of discrimination is achievedsuch that an assay generates an accurate (within the tolerance desiredfor the assay) and reproducible result. Nevertheless, aided by no morethan routine experimentation and the disclosure provided herein, thoseof skill in the art will easily be able to determine suitablehybridization conditions for performing assays utilizing the methods andcompositions described herein.

Probing Nucleobase Sequence:

The probing nucleobase sequence of probe is the specific sequencerecognition portion of the construct. Therefore, the probing nucleobasesequence is an aggregate nucleobase sequence of the probe that isdesigned to hybridize to a specific target sequence of interest in asample. By aggregate nucleobase sequence, we refer to the totality ofthe nucleobase subunits that bind to the target sequence without regardto whether or not they comprise one or more linkages atypical to thebackbone of the polymer (e.g. two segments of continuous nucleobasesequence containing subunits separated by a linker). The target sequencecan be a sequence that identifies a gene such as for example, theCyclinD1 gene, the c-MYC gene, the EGFR gene, the TEL gene, the E2Agene, the BCR gene, the IGH gene, the IGL gene or the IGK gene.

Advantages of the Present Invention:

The non-nucleotide probes of this invention can be chemicallysynthesized and purified in a manner that provides for low cost andproper characterization. Consequently, the unlabeled non-nucleotideprobes (e.g. peptide nucleic acid oligomers) can be individuallyprepared, characterized and quantitated before preparing a mixture ofprobes for a blocking application. Hence, the mixture of probes itselfcan therefore be more carefully controlled, characterized and reproducedthan are the Cot1 DNA probes of the prior art. Since one possibleapplication for such a the mixture of probes is their possible use in adiagnostic assay, the ability to more easily characterize and reproduce,in a cost effective manner, the exact composition of the mixture fromone batch to the next is potentially advantageous.

Applications for the Present Invention:

The suppression methods described herein can be useful in analyzingcells for the occurrence of chromosomes, chromosome fragments, genes, orchromosome aberrations (e.g. translocations, deletions, amplifications)associated with a condition or disease. Any method that can detect,identify and/or quantify selected target genomic nucleic acid inmetaphase spreads, interphase nuclei, tissue sections, and extracted DNAfrom these cells can potentially take advantage of the present method asa substitute for the conventional Cot-1 DNA blocking. These methodsinclude, but are not limited to, CISH (chromogen in situ hybridization),FISH, multi-color FISH, Fiber-FISH, CGH, chromosome paints and theanalysis of BAC clones and arrays.

TABLE 1 Seq. Id. Nucleobase Sequence No. GGCGGGCGGAGGCCGGGCGCGGTGGCTCA 1CGCCTGTAATCCCAGCACTTTGGGAGGCC GAGGCGGGCGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAA ACCCCGTCTCTACTAAAAATACAAAAATTAGCCGGGCGTGGTGGCGCGCGCCTGTART CCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCGGAGGTTG CAGTGAGCCGAGATCGCGCCACTGCACTCCAGCCTGGGCRACAAGAGCGARACTCCGT CTCAAAAAAAA TTTTTTTTGAGACGGAGTYTCGCTCTTGT2 YGCCCAGGCTGGAGTGCAGTGGCGCGATC TCGGCTCACTGCAACCTCCGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCC GAGTAGCTGGGAYTACAGGCGCGCGCCACCACGCCCGGCTAATTTTTGTATTTTTAGT AGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGAT CCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCGGC CTCCGCCCGCC GGCCGGGCGCGGTGGCT 3GCTGGGATTACAGGCGTG 4 GGGAGGCCGAGGCGGG 5 GCCAGGCTGGTCTCGAACTCC 6GAAACCCCGTCTCTACTAAAA 7 GCCGGGCGTGGTGGCG 8 TAGCTGGGATTACAGGCG 9GGGAGGCTGAGGCAGGA 10 CCTCCCGGGTTCAAGCGATTC 11 TTGCAGTGAGCCGAGAT 12TGCACTCCAGCCTGGGCGACA 13 **TT(k)TTTTT(k)TTTLysOLysOTTT (k)TTTTT(k)TTAGCCACCGCGCCCGGCC 15 CACGCCTGTAATCCCAGC 16 CCCGCCTCGGCCTCCC 17GGAGTTCGAGACCAGCCTGGC 18 TTTTAGTAGAGACGGGGTTTC 19 CGCCACCACGCCCGGC 20CGCCTGTAATCCCAGCTA 21 TCCTGCCTCAGCCTCCC 22 GAATCGCTTGAACCCGGGAGG 23ATCTCGGCTCACTGCAA 24 TGTCGCCCAGGCTGGAGTGCA 25AA(k)AAAAA(k)AAA-Lys-O-Lys-O- AAA(k)AAAAA(k)AA Note: k = D-lysine; Lys =L-lysine; O = 8-amino-3,6-dioxaoctanoic acid;

II. Embodiments of the Invention

Generally, this invention pertains to methods, kits, non-nucleotideprobes as well as other compositions for the suppression of binding ofdetectable nucleic acid probes to undesired nucleotide sequences ofgenomic nucleic acid in assays designed to determine target genomicnucleic acid. In many cases, the most problematic undesired nucleotidesequences are those known in the art as randomly distributed repeatsequences, which include, but are not limited to, Alu-repeats,Kpn-repeats, di-nucleotide repeats, tri-nucleotide repeats,tetra-nucleotide repeats, penta-nucleotide repeats, hexa-nucleotiderepeats, SINEs and LINEs. These are referred to as randomly distributedrepeat sequences since they are not prevalent in any particular sectionof the genetic material, such as in a centromere or telomere region, butrather are randomly distributed within all of the chromosomes of anorganism.

Non-Nucleotide Probes:

In one embodiment, this invention pertains to a non-nucleotide probe ofat least sixteen nucleobase containing subunits in length having anaggregate nucleobase sequence that is at least eighty percent homologousto a sixteen nucleotide segment of randomly distributed repeat sequenceof genomic nucleic acid. The segment of randomly distributed repeatsequence can be a SINE or LINE. SINEs and LINEs can be selected from thegroup consisting of: Alu-repeats, Kpn-repeats, di-nucleotide repeats,tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotiderepeats and hexa-nucleotide repeats.

The nucleobase sequence of the non-nucleotide probe can be chosen to besubstantially, or completely, homologous to a fraction, or part, ofeither: (i) a known unit repeat of a Alu-repeat sequence; or (ii) aconsensus sequence of a unit repeat of a known Alu-repeat sequence. Forexample, the segment can contain at least ten consecutive nucleobasesthat are at least eighty percent homologous to the unit repeat consensusAlu-repeat sequences selected from the group consisting of: Seq. Id. No.1 and Seq. Id. No. 2 (See Table 1). The ten consecutive nucleobases canbe at least ninety percent homologous to the identified Alu-repeatconsensus sequences or they can be exactly homologous to the identifiedAlu-repeat consensus sequences. The non-nucleotide probe can be fromabout 16 to about 50 nucleobase containing subunits in length. Thenon-nucleotide probe can be a peptide nucleic acid oligomer.

In another embodiment, this invention pertains to a non-nucleotide probecontaining an aggregate nucleobase sequence of at least ten consecutivenucleobases that is at least eighty percent homologous to the sequencesselected from the group consisting of: Seq. Id. No. 3, Seq. Id. No. 4,Seq. Id. No. 5, Seq. Id. No. 6, Seq. Id. No. 7, Seq. Id. No. 8, Seq. Id.No. 9, Seq. Id. No. 10, Seq. Id. No. 11, Seq. Id. No. 12, Seq. Id. No.13, Seq. Id. No. 14, Seq. Id. No. 15, Seq. Id. No. 16, Seq. Id. No. 17,Seq. Id. No. 18, Seq. Id. No. 19, Seq. Id. No. 20, Seq. Id. No. 21, Seq.Id. No. 22, Seq. Id. No. 23, Seq. Id. No. 24, Seq. Id. No. 25 and Seq.Id. No. 26. Certain of these particular sequences have been determinedto be highly effective at suppressing the binding of a detectablenucleic acid probe to undesired chromosomes in an assay for detectingthe HER-2 or MLL target nucleic acid sequences in genomic nucleic acid(See: Examples 4 and 5). Complementary sequences to the testednucleobase sequences are included because these non-nucleotide probesare directed to genomic nucleic acid that is typically present in doublestranded form. Hence, the target sequences for these probe sequences, aswell as their complements, can be present in samples containing thecomplementary strands of genomic nucleic acid.

The ten consecutive nucleobases can be either: (i) at least ninetypercent homologous to the identified sequences; or (ii) exactlyhomologous to the identified sequences. The probe can be identical innucleobase sequence to any one of the identified sequences. Thenon-nucleotide probe can be from about 10 to about 50 nucleobasecontaining subunits in length. The non-nucleotide probe can be a peptidenucleic acid oligomer.

The aforementioned non-nucleotide probes will either be labeled orunlabeled. The exact configuration of the probe will usually depend uponits intended use. For example, if the probe is being screened in orderto determine whether or not it may be useful in binding to undesiredsequence in genomic nucleic acid, as described in more detail below, itis likely to be labeled. Conversely, if the probe is being used in amethod to suppress the binding of detectable nucleic acid probe toundesired sequence in genomic nucleic acid, as described in more detailbelow, it is likely to be unlabeled.

Probe Mixtures:

In still another embodiment, this invention pertains to a mixture of twoor more non-nucleotide probes wherein each probe contains an aggregatenucleobase sequence that is at least eighty percent homologous to asixteen nucleotide segment of randomly distributed repeat sequence ofgenomic nucleic acid. The mixture can comprise from about 5 to about 50probes of different nucleobase sequence. The mixture can comprise fromabout 10 to about 25 probes of different nucleobase sequence. Theindividual probes of the mixture can comprise from about 10 to about 50nucleobase containing subunits in length.

Any of the aforementioned non-nucleotide probes can be suitable for usein the non-nucleotide probe mixture. The non-nucleotide probes can bepeptide nucleic acid oligomers. The mixture of probes can furthercomprise one or more detectable nucleic acid probes. The mixture ofprobes can further comprise genomic nucleic acid of a sample to betested.

Probes & Probe Mixtures:

The non-nucleotide probes or mixture of non-nucleotide probes of thisinvention can be manufacture and purified using conventional methods,including without limitation, those previously described herein. Theymay be provided, used, handled and/or dispensed either as a dry(lyophilized) powder, dissolved or suspended in a solvent or mixed witha dry carrier. Mixtures of non-nucleotide probes can be, but are notnecessarily, produced by first manufacturing and purifying the componentprobes followed by a step of mixing the probes in the desired ratio tothereby produce the mixture. Suitable solvents and carriers for thenon-nucleotide probes are known in the art and include, withoutlimitation, solutions of water that optionally comprise an organicmodifier, such as N,N′-dimethylformamide (DMF) or 1-Methyl-2-pyrrolidone(NMP), and/or buffer.

Hybrid Compositions:

In yet another embodiment, this invention pertains to a compositioncomprising genomic nucleic acid containing one or more segments ofrandomly distributed repeat sequence selected from the group consistingof: SINEs and LINEs. The SINEs and LINEs can be selected from the groupconsisting of: Alu-repeats, Kpn-repeats, di-nucleotide repeats,tri-nucleotide repeats, tetra-nucleotide repeats, penta-nucleotiderepeats and hexa-nucleotide repeats. The one or more segments ofrandomly distributed repeat sequence can be a fraction, or part, of aunit repeat of either: i) an Alu-repeat sequence; or ii) a consensussequence of a Alu-repeat sequence. The composition further comprises twoor more non-nucleotide probes of different nucleobase sequencehybridized to at least a fraction, or part, of the one or more segmentsof randomly distributed repeat sequence of the genomic nucleic acid.Hence, the composition is the hybrid of the segment of randomlydistributed repeat sequence and the two or more non-nucleotide probes.The non-nucleotide probes can suppress the binding of detectable nucleicacid probe to the randomly distributed repeat sequence of the genomicnucleic acid.

Any of the aforementioned non-nucleotide probes, including preferredembodiments thereof, are suitable for use in producing the hybrid. Thenon-nucleotide probes can be peptide nucleic acid oligomers.

The genomic nucleic acid of the hybrid can comprise complementarystrands of randomly distributed repeat sequence. The genomic nucleicacid can be contained within a fixed tissue or a cell. The genomicnucleic acid can be contained within metaphase spreads, interphasenuclei or the nuclei of paraffin embedded tissue material or frozentissue sections. The nucleic acid can also be extracted from any of theaforementioned samples.

An illustration of an exemplary construct of this hybrid is found inFIG. 1. The design of the aforementioned construct is intended to allowthe non-nucleotide probes to hybridize to the complementary strands ofgenomic nucleic acid in such a manner as to cover as much as possible ofone or the other of the complementary strands of the genomic nucleicacid. This approach can also serve to keep the strands separated. Theability to lock the complementary strands of nucleic acid into an openconformation under hybridization conditions is surprising in view of theteachings of Perry-O'Keefe et al. (Proc. Natl. Acad. Sci. USA, 93:14670-14675 (1996)) who specifically teach that DNA reannealing willexpel bound PNA probe that is much shorter. Because the PNA oligomersare short, as compared with the complementary strands of genomic nucleicacid, the PNA oligomers are expected to be expelled and thereby shouldnot act to block the hybridization of the longer detectable nucleic acidprobes to these undesired sequences.

Because it may be desirable to provide a mixture of non-nucleotideprobes in the same container as the detectable nucleic acid probes, andbecause the detectable nucleic acid probes can possess segments ofsequence that are derived from the randomly distributed repeatsequences, this invention is still further directed to a compositioncomprising a detectable nucleic acid probe of at least 100 bp that hasbeen derived from genomic nucleic acid and that contains one or moresegments of randomly distributed repeat sequence selected from the groupconsisting of: SINEs and LINEs. The SINEs and LINEs can be selected fromthe group consisting of: Alu-repeats, Kpn-repeats, di-nucleotiderepeats, tri-nucleotide repeats, tetra-nucleotide repeats,penta-nucleotide repeats and hexa nucleotide repeats. The compositionfurther comprises two or more non-nucleotide probes of differentnucleobase sequence hybridized to at least a fraction of the one or moresegments of randomly distributed repeat sequence of the detectablenucleic acid probe. Hence, the composition is the hybrid of thedetectable nucleic acid probe hybridized to the two or morenon-nucleotide probes.

Any of the aforementioned non-nucleotide probes can be suitable forproducing the hybrid. The non-nucleotide probes can be peptide nucleicacid oligomers.

Method for the Suppression of Undesired Detectable Probe Binding:

In still another embodiment, this invention is directed to a method forsuppressing the binding of one or more detectable nucleic acid probes,that are greater than 100 bp and that have been derived from genomicnucleic acid, to one or more undesired sequences in an assay fordetermining target genomic nucleic acid of a sample. The methodcomprises contacting the sample with a mixture of two or morenon-nucleotide probes wherein each probe contains an aggregatenucleobase sequence that is at least eighty percent homologous to asegment of randomly distributed repeat sequence of genomic nucleic acid.According to the method, the sample is also contacted with the one ormore detectable nucleic acid probes. The target genomic nucleic acid ofthe sample can then be determined by determining the hybridization ofthe one or more detectable nucleic acid probes to the target genomicnucleic acid of the sample wherein the presence, absence or amount ofhybridization of the detectable nucleic acid probe to the target genomicnucleic acid can be representative of the presence, absence or amount oftarget genomic nucleic acid in the sample.

The randomly distributed repeat sequences can be selected from the groupconsisting of: SINEs and LINES. The SINEs and LINEs can be selected fromthe group consisting of; Alu-repeats, Kpn-repeats, di-nucleotiderepeats, tri-nucleotide repeats, tetra-nucleotide repeats,penta-nucleotide repeats and hexa-nucleotide repeats. The nucleobasesequence of SINEs and LINEs can be used as the basis for producingprobes that are suitable to suppress undesired binding since Applicantshave shown that blocking of the Alu-repeat sequences are particularlyuseful in lowering the background signal that is otherwise present insuch assays (See: Example 4).

According to the method, the nucleobase sequence of the non-nucleotideprobe can be selected to be at least eighty percent homologous to a partof a consensus sequence of a randomly distributed repeat sequence. Thenucleobase sequence of the non-nucleotide probe can contain a segment ofat least ten consecutive nucleobases that is at least eighty percenthomologous to a fraction, or part, of the consensus unit repeatAlu-repeat sequences selected from the group consisting of: Seq. Id. No.1 and Seq. Id. No. 2. The ten consecutive nucleobases can be at leastninety percent homologous to the identified consensus sequences. The tenconsecutive nucleobases can be exactly homologous to the identifiedconsensus sequences.

Any of the aforementioned non-nucleotide probes can be suitable for usein producing the hybrid that suppresses the binding of the detectablenucleic acid probes to the undesired sequence. The two or morenon-nucleotide probes can be about 10 to about 50 nucleobase containingsubunits in length. The non-nucleotide probes can be peptide nucleicacid oligomers.

According to the method, the genomic nucleic acid can comprisecomplementary strands of randomly distributed repeat sequence. Thegenomic nucleic acid can be contained in a fixed tissue or a cell. Thegenomic nucleic acid can be contained in metaphase spreads, interphasenuclei or the nuclei of paraffin embedded tissue material or frozentissue sections. The nucleic acid can also be extracted from the cellsor tissues.

Genomic Arrays:

(i) Array Comparative Genomic Hybridization (Array CGH)

Chromosomal comparative genomic hybridization (CGH) allows acomprehensive analysis of multiple DNA gains and losses in entiregenomes within a single experiment. Genomic DNA from the tissue to beinvestigated, such as fresh or paraffin-embedded tumor tissue and normalreference DNA, are differentially labeled and simultaneously hybridizedin situ to normal metaphase chromosomes. Array-based Comparative GenomicHybridization (array CGH) provides a higher-resolution and morequantitative alternative to chromosome CGH for the assessment of genomiccopy number abnormalities. Instead of hybridizing to individualchromosomes (as occurs in a ISH or FISH assay) with array CGH copynumber abnormalities are mapped onto arrays of cloned DNA sequences suchas P1s, BACs or cDNAs with the fluorescence ratios at the arrayed DNAelements providing a locus-by-locus measure of DNA copy-numbervariation. The basic assumption of a CGH experiment is that the ratio ofthe binding of test and control DNA is proportional to the ratio of theconcentrations of sequences in the two samples.

(ii) Methodology of CGH

For CGH or array CGH, whole-genomic DNA is isolated from a tumor bystandard extraction protocols. Control or reference DNA is isolated froman individual who has either a normal 46, XX karyotype or a normal 46,XY karyotype. There is also a sample that is to be analyzed bycomparison to the control or reference DNA. The control and sample DNAthat has been extracted from the two genomes is differentially labeled(for example fluorescein conjugated to dUTP for the tumor genome and Cy3conjugated to dUTP for the normal genome). The sample and control DNAsamples are combined, and an excess of unlabelled blocking reagent (e.g.a mixture of non-nucleic acid probes as described herein) is added intothe hybridization mixture, to suppress the repetitive sequences that arepresent in both genomes. The blocking reagent is useful becausehybridization of the repetitive DNA would impair the evaluation of theunique sequences that are either over represented or underrepresented inthe sample genome. This probe mixture is hybridized to normal humanreference metaphase chromosomes or arrays of cloned DNA sequences in thecase of array CGH. The relative color intensities of the twofluorochromes reflect DNA copy-number alterations in the tumor genome.In this way it is possible to determine whether or not the sample DNA isnormal (where the color intensities of the two fluorophores is the same)or abnormal (where the color intensities of the two samples differs).The degree of difference in the color intensities for the twofluorophores can also a measure of the severity or identity of a diseasestate.

(iii) Array Based Embodiments of the Invention

Thus, in yet another embodiment, this invention pertains to comparing asample of genomic nucleic acid with that of a control sample using agenomic nucleic acid reference array. The method comprises providing asample of genomic nucleic acid to be tested, providing a control ofgenomic nucleic acid, wherein the control and the sample aredifferentially labeled. The method further comprises providing a genomicnucleic acid reference array, and providing a mixture of two or morenon-nucleotide probes wherein each probe contains an aggregatenucleobase sequence that is at least eighty percent homologous to asixteen nucleotide segment of randomly distributed repeat sequence ofgenomic nucleic acid. The method further comprises treating the sampleand control genomic nucleic acid, the array or both the sample andcontrol genomic nucleic acid and the array with the mixture ofnon-nucleotide probes under suitable hybridization conditions. The arrayis then contacted with the treated mixture of sample and control genomicnucleic acid under suitable hybridization conditions. The intensities ofthe signals from the differential labels on the array, caused byhybridization of the probes to genomic nucleic acid, are then comparedto thereby determine one or more variations in copy numbers of sequencesin the sample as compared with the relative copy numbers ofsubstantially identical sequences in the control.

Method for Determining Non-Nucleotide Probes:

In still another embodiment, this invention is directed to a method fordetermining non-nucleotide probes that hybridize to randomly distributedrepeat sequences and that are suitable for suppressing the binding of adetectable nucleic acid probe, that is greater than 100 bp in length andthat is derived from genomic nucleic acid, to one or more undesirablesequences in an assay for determining target genomic nucleic acid of asample. The method comprises designing possible nucleobase sequences ofnon-nucleotide probes using sequence alignment of available sequencedata for randomly distributed repeat sequences and then preparinglabeled non-nucleotide probes having said possible nucleobase sequences.According to the method, genomic nucleic acid of a sample that containsthe target genomic nucleic acid is treated with the labelednon-nucleotide probes under suitable hybridization conditions. Therelative signal of the hybridized labeled probes of the many differentpossible nucleobase sequences is then determined. Based upon the signalintensity data, the probe or probes that exhibit the strongest signal,as a result of binding to the genomic nucleic acid, are selected andtested to thereby determine whether or not they are suitable forsuppressing the binding of a detectable nucleic acid probe of greaterthan 100 bp in length that is derived from genomic nucleic acid to oneor more non-target sequences in an assay for determining target genomicnucleic acid of a sample. In order to test the one or more selectednon-nucleotide probes, each probe can be re-synthesized in unlabeledform and then tested using the method for suppressing the binding ofdetectable probes to undesired sequences as described above. Oncetested, the best probes can be used to produce a mixture that can beused in an assay to suppress the binding of detectable nucleic acidprobes to undesired target sequence. The non-nucleotide probes can bepeptide nucleic acid oligomers.

Those of skill in the art will appreciate that sequence alignment is aprocess that can be performed using a database of sequence informationthat is analyzed using a computer and software designed to analyze thedatabase in accordance with a particular set of input parameters. In thecontext of the present invention, the sequence of available randomlydistributed repeat sequences would be provided in the database. Fromthis database, potential probe sequences can be selected in accordancewith the output provided from the software program operating by computeranalysis of the database in view of the input parameters. The inputparameters can be directed toward providing a consensus sequence wherethere are sequence variations known to exist among the various randomlydistributed repeats sequences. Whether using a known randomlydistributed repeat sequence, or a consensus sequence as can be seen fromthe data in Example 3, one should broadly choose all possible nucleobasesequences and then screen the candidates since small variations of oneor two nucleobases can substantially alter the hybridization performanceof the non-nucleotide probes.

As illustrated by the Examples of this specification, this method hasbeen shown to be very useful in selecting probes that can be effectivelyused to “block” or suppress the binding of detectable nucleic acidprobes to undesired sequences in genomic nucleic acid. Although thismethod has been shown to be effective with respect to randomlydistributed repeat sequences, it is anticipated that said method can beequally useful in the design of probes or probe mixtures that aid in thesuppression of binding of detectable probe to other undesired sequencesof genomic nucleic acid. In order to extend the aforementioned method toother problematic sequences it is only required that one identify apotentially problematic sequence or sequences from which potentiallyuseful blocking probes can be generated for screening purposes inaccordance with the aforementioned method.

Kits:

In still another embodiment, this invention is directed to a reagent kitcomprising a mixture of two or more non-nucleotide probes containing atleast sixteen consecutive nucleobases that are at least eighty percenthomologous to a fraction of the unit repeat Alu-repeat consensussequence selected from the group consisting of: Seq. Id. No. 1 or Seq.Id. No. 2. The kit further comprises one or more other reagents,compositions and or instructions suitable for performing an assay tothereby determine genomic nucleic acid of a sample. For example, thereagent kit can further comprise one or more detectable nucleic acidprobes of greater than 100 bp in length and that are derived fromgenomic nucleic acid. The one or more detectable nucleic acid probes canbe provided in the container that contains the mixture of two or morenon-nucleotide probes.

In yet still another embodiment, this invention is directed to a kitcomprising a mixture of two or more non-nucleotide probes wherein atleast one probe contains a segment of at least ten consecutivenucleobases that are at least eighty percent homologous to theAlu-repeat sequences selected from the group consisting of: Seq. Id. No.3, Seq. Id. No. 4, Seq. Id. No. 5, Seq. Id. No. 6, Seq. Id. No. 7, Seq.Id. No. 8, Seq. Id. No. 9, Seq. Id. No. 10, Seq. Id. No. 11, Seq. Id.No. 12, Seq. Id. No. 13, Seq. Id. No. 14, Seq. Id. No. 15, Seq. Id. No.16, Seq. Id. No. 17, Seq. Id. No. 18, Seq. Id. No. 19, Seq. Id. No. 20,Seq. Id. No. 21, Seq. Id. No. 22, Seq. Id. No. 23, Seq. Id. No. 24, Seq.Id. No. 25 and Seq. Id. No. 26. The kit further comprises at least oneother reagent, composition and/or set of instructions for performing aassay to thereby determine genomic nucleic acid of a sample. Forexample, the kit can further comprise one or more detectable nucleicacid probes of greater than 100 bp in length and that are derived fromgenomic nucleic acid. The one or more detectable nucleic acid probes canbe provided in the container that contains the mixture of two or morenon-nucleotide probes.

Said kits are particularly useful since they provide reagents suitableto perform a specific type of assay in convenient packaging therebyeliminating the need to devise an assay and then prepare the necessaryreagents. The kits can provide the necessary reagents to perform anassay for detecting the HER-2 or MLL target sequence in a samplecontaining human genomic nucleic acid.

Having described the preferred embodiments of the invention, it will nowbecome apparent to one of skill in the art that other embodimentsincorporating the concepts described herein may be used. It is felt,therefore, that these embodiments should not be limited to disclosedembodiments but rather should be limited only by the spirit and scope ofthe following claims.

EXAMPLES

This invention is now illustrated by the following examples that are notintended to be limiting in any way.

General Information on PNA Oligomer Synthesis

All PNA Oligomers were prepared from commercial reagents andinstrumentation obtained from Applied Biosystems, Foster City, Calif.using manufacturer published procedures, other well-known procedures orthose disclosed in U.S. Pat. Nos. 5,888,733, 5,985,563, 6,110,676,6280,946, 6,287,772, 6,326,479, 6,355,421, 6,361,942 and 6,441,152 (allof which are herein incorporated by reference). All PNA oligomers werepurified by reversed-phase high performance liquid chromatography usingwell-known methods. Table 2 lists PNA oligomers used in Examples 4-5,described below.

TABLE 2 Probe Seq. No. Label Position N-term Sequence C-term Id. No. 1 #CAGGCCGGGTGCAGTGGC 35 2 fluore # Lys (Flu) CAGGCCGGGTGCAGTGGC scein 3 9-25 GGCCGGGCGCGGTGGCT  3 4 fluore  9-25 Lys (Flu) GGCCGGGCGCGGTGGCTscein 5  9-25 EE GGCCGGGCGCGGTGGCT EE 6 fluore  9-25 Flu-OEEGGCCGGGCGCGGTGGCT EE scein 7 fluore  9-25 FluOLysLys GGCCGGGCGCGGTGGCTLysLys scein 8 biotin 10-24 Bio-OEE GCCGGGCGYGGTGGC EE 36 9 10-24 EEGCCGGGCGYGGTGGC EE 10 fluore 10-24 Flu-OEE GCCGGGCGYGGTGGC EE scein 1126-43 GCTGGGATTACAGGCGTG  4 12 fluore 26-43 Lys (Flu) GCTGGGATTACAGGCGTGscein 13 26-43 Lys-Lys GCTGGGATTACAGGCGTG Lys-Lys 14 26-43 EEGCTGGGATTACAGGCGTG EE 15 fluore 26-43 Flu-OEE GCTGGGATTACAGGCGTG EEscein 16 biotin 28-43 Bio-OEE GCTGGGAYTACAGGCG EE 37 17 32-48TGTAATCCCAGCACTTT 38 18 fluore 32-48 Lys (Flu) TGTAATCCCAGCACTTT scein19 fluore 42-56 Lys (Flu)- GCACTTTGGGAGGCC 39 scein Lys (Flu) 20 fluore42-56 Lys (Flu)- GGCCTCCCAAAGTGC 40 scein Lys (Flu) 21 biotin 49-54Bio-OEE CCTCCC-EOE-CCCTCC EE 22 biotin 49-63 Bio-OEE GGGAGGCYGAGGCGG EE42 23 49-64 GGGAGGCCGAGGCGGG  5 24 fluore 49-64 Flu-O GGGAGGCCGAGGCGGGscein 25 fluore 49-64 FluOLysLys GGGAGGCCGAGGCGGG LysLys scein 26 49-64EE GGGAGGCCGAGGCGGG EE 27 fluore 49-64 Flu-OEE GGGAGGCCGAGGCGGG EE scein28 # ACTTTGGGAGGAAGATCACC 43 29 fluore # Flu-Lys ACTTTGGGAGGAAGATCACCscein 30 70-84 CACCTGAGGTCAGGA 44 31 fluore 70-84 Flu-OE CACCTGAGGTCAGGAE scein 32  82-102 GCCAGGATGGTCTCGATCTCC 27 33 fluore  82-102 Lys (Flu)GCCAGGATGGTCTCGATCTCC scein 34 82-102 GCCAGGCTGGTCTCGAACTCC  6 35 fluore82-102 Lys (Flu) GCCAGGCTGGTCTCGAACTCC scein 36  82-102 Lys-LysGCCAGGCTGGTCTCGAACTCC Lys-Lys 37  82-102 EE GCCAGGCTGGTCTCGAACTCC EE 38fluore  82-102 Flu-OEE GCCAGGCTGGTCTCGAACTCC EE scein 39  98-113TGGCCAACATGGTGA 45 40 fluore  98-113 Flu-OE TGGCCAACATGGTGA E scein 41112-132 GAAACCCCGTCTCTACTAAAA  7 42 fluore ll2-132 Lys (Flu)GAAACCCCGTCTCTACTAAAA scein 43 112-132 Lys GAAACCCCGTCTCTACTAAAA Lys 44112-132 Lys-Lys GAAACCCCGTCTCTACTAAAA Lys-Lys 45 112-132 EEGAAACCCCGTCTCTACTAAAA EE 46 fluore 112-132 Flu-OEE GAAACCCCGTCTCTACTAAAAEE scein 47 145-160 GCCGGGCGTGGTGGCG  8 48 fluore 145-160 Lys (Flu)GCCGGGCGTGGTGGCG scein 49 145-160 EE GCCGGGCGTGGTGGCG EE 50 fluore145-160 Flu-OEE GCCGGGCGTGGTGGCG EE scein 51 fluore 145-160 FluOLysLysGCCGGGCGTGGTGGCG LysLys scein 52 163-180 TAGCTGGGATTACAGGCG Lys  9 53fluore 163-180 Lys (Flu) TAGCTGGGATTACAGGCG Lys scein 54 163-180 Lys-LysTAGCTGGGATTACAGGCG Lys-Lys 55 163-180 EE TAGCTGGGATTACAGGCG EE 56 fluorel63-180 Flu-OEE TAGCTGGGATTACAGGCG EE scein 57 184-200 GGGAGGCTGAGGCAGGALys 10 58 fluore 184-200 Lys (Flu) GGGAGGCTGAGGCAGGA Lys scein 59184-200 Lys-Lys GGGAGGCTGAGGCAGGA Lys-Lys 60 184-200 EEGGGAGGCTGAGGCAGGA EE 61 fluore 184-200 Flu-OEE GGGAGGCTGAGGCAGGA EEscein 62 186-200 GAGGCTGAGGCAGGA 46 63 fluore 186-200 Lys (Flu)GAGGCTGAGGCAGGA scein 64 201-221 CCTCCCGGGTTCACGCCATTC 47 65 fluore201-221 Lys (Flu) CCTCCCGGGTTCACGCCATTC scein 66 201-221CCTCCCGGGTTCAAGCGATTC Lys 11 67 fluore 201-221 Lys (Flu)CCTCCCGGGTTCAAGCGATTC Lys scein 68 201-221 EE CCTCCCGGGTTCAAGCGATTC EE69 fluore 201-221 Flu-OEE CCTCCCGGGTTCAAGCGATTC EE scein 70 fluore201-221 FluOLysLys CCTCCCGGGTTCAAGCGATTC LysLys scein 71 228-244 LysTTGCAGTGAGCCGAGAT 12 72 fluore 228-244 Lys (Flu) TTGCAGTGAGCCGAGAT scein73 fluore 228-244 FluOLysLys TTGCAGTGAGCCGAGAT LysLys scein 74 228-244EE TTGCAGTGAGCCGAGAT EE 75 fluore 228-244 Flu-OEE TTGCAGTGAGCCGAGAT EEscein 76 fluore 228-244 Flu-OPP TTGCAGTGAGCCGAGAT PP scein 77 fluore228-244 Flu-OOO TTGCAGTGAGCCGAGAT OO scein 78 fluore 228-244 Flu-TTGCAGTGAGCCGAGAT GluGlu scein OGluGlu 79 fluore 228-244 Lys (Flu)DUCUCGGCUCDCUGCDD Lys 48 scein 80 fluore 228-244 Lys (Flu)UUGCDGUGDGCCGDGDU Lys 49 scein 81 249-273 CCACTGCACTCCAGCCTGG 50 GCGACA82 fluore 249-273 Lys (Flu) CCACTGCACTCCAGCCTGG scein GCGACA 83 253-269Lys TGCACTCCAGCCTGGGC 51 84 fluore 253-269 Lys (Flu) TGCACTCCAGCCTGGGCscein 85 253-273 TGCACTCCAGCCTGGGCGACA 13 86 fluore 253-273 Lys (Flu)TGCACTCCAGCCTGGGCGACA scein 87 253-273 Lys-Lys TGCACTCCAGCCTGGGCGACALys-Lys 88 253-273 EE TGCACTCCAGCCTGGGCGACA EE 89 fluore 253-273 Flu-OEETGCACTCCAGCCTGGGCGACA EE scein 90 # TTTGAGACAGAGTCTCGC 52 91 fluore #Lys (Flu) TTTGAGACAGAGTCTCGC scein 92 275-294 TTTGAGACGGAGTCTCGCTC Lys53 93 fluore 275-294 Lys (Flu) TTTGAGACGGAGTCTCGCTC Lys scein 94 fluore292-298 FluLys (Flu)OO TTTTTTT-O-Lys-O-Lys-O- Lys scein TTTTTTT 95292-298 FluLysOO TTkTTTTT-O-Lys-O-Lys- Lys O-TTkTTTTT 96 fluore 292-298FluLys (Flu)OO TTkTTTTT-O-Lys-O-Lys- Lys scein O-TTkTTTTT 97 PolyA LysTTkTTTTTkTTTLysOLysOTT Lys tail TkTTTTTkTT 98 fluore PolyA FluOLysTTkTTTTTkTTTLysOLysOTT Lys scein tail TkTTTTTkTT 99 fluore PolyA FluOTTkTTTTTkTTTOOOTTTkTTT scein tail TTkTT fluorescein or Flu =5(6)-carboxyfluorescein; Lys = the amino acid L-lysine, k = the aminoacid D-lysine; 0 8-amino-3,6-dioxaoctanoic acid; E = the modificationresulting from use of compound 4 as described in Gildea et al., Tett.Lett. 39: 7255-7258 (1998)), Bio = biotin; Glu = the amino acid glutamicacid; d = the product of using piperazine-N,N′ diacetic acid-mono(2-Boc-aminoethylamide) as a monomer; #: alu sequence, but not consensussequence; *: score not comparable to directly labeled oligomersGeneral Information on Nucleic Acid Oligomers

Those of skill in the art will appreciate that detectable nuclei acidprobes can be produced by selection of a clone covering the desiredregion of interest from a public library of clones (e.g. Resourcezemtrumin Deutchen Humangenomprojekt, RZPD). The DNA from such a clone can thenbe cultured within a host organism, extracted from the host, purified,and labeled. To amplify a specific DNA sequence by cloning, the DNA canbe inserted into a vector and both insert and vector were amplifiedinside appropriate host cells. The amplified DNA can then be extracted.Commonly used vectors include bacterial plasmids, cosmids, PACs, BACs,and YACs, all of which are well known to one or ordinary skill in theart.

The purified DNA is then labeled by the Nick translation. The Nicktranslation reaction employs two enzymes, Dnase I which produces the“nicks” in the double-stranded DNA and DNA polymerase, which incorporatelabeled nucleotides along both strands of the DNA duplex. It will beappreciated that using no more that routine experimentation andinformation known to those of ordinary skill in the art that any knownlabeling method can be used for labeling the nucleic acid probes used inthe embodiments and/or description of this invention.

In Examples 4 and 5, fluorescence labeled COS or PAC based DNA probeswere produced by culturing the COS or PAC containing E. coli andharvesting the human DNA from the cultures. The COS or PAC DNA waspurified using Qiagen large construct kit (Qiagen, Kebolab A/S,Copenhagen, Denmark). The clones were checked by restriction enzymedigestion. From each of the clones, the DNA was labeled by conventionalNick translation using a monomer labeled with either fluorescein, Cy3,or Rhodamine, as appropriate. The Nick translation was performed usingwell known methods.

General Information on Preferred Method for Cytogenetic Preparations:

Metaphase spreads and interphase nuclei were prepared from humanperipheral blood lymphocytes. A blood sample of 0.5 mL was added to 10mL culture medium (RPMI 1640 medium supplemented with 20% fetal calfserum, 2 mM Glutamine, 100 U/mL Penicillin/Streptomycin, 1%Phytohemagglutine, and 50 U/mL Heparin) and cultured for 72 hours at 37°C. For metaphase arrest the culture was incubated with 0.1 μg/mLColcemid (Gibco, BRL) for 90 min at 37° C. The culture was thencentrifuged at 500×g for 10 min. The supernatant was removed leaving 1mL for resuspension in 8 mL 60 mM KCl. After incubation for 30 min atroom temperature (RT), the cells were pre-fixated by adding 1 mL freshlymade Fixative (3+1 v/v methanol/acetic acid) on top of the hypotonicsuspension, and mixed carefully by turning the tube. After 10 min at RT,the suspension was pelleted by centrifugation at 500×g for 10 min. Thesupernatant was then removed leaving 1 mL that was resuspended in 10 mLFixative added slowly with gentle agitation. The fixation was repeatedtwice with at 10 min incubation at RT between each fixation. After thethird fixation the cells were resuspended in 1 mL Fixative. The cellswere then dropped onto wet microscopic slides that have been cleaned indetergent and rinsed with water. The slides are left to air dry andstored at −20° C. until hybridization.

Example 1: Design of Non-Nucleotide Probes Directed Towards RepetitiveSequences

For this Example, the nucleobase sequences of non-target hybridizationprobes directed toward Alu repeat consensus sequence were designed. Itwas envisioned that the probe sequences that would be best forsuppressing the binding of detectable nucleic acid probes, generatedfrom nick translation of cosmid nucleic acid, would be those nucleobasesequences possessing shared repeated sequences of Alu repeat sequencethat are most prevalent in genomic nucleic acid. Hence, a consensussequence of known Alu-repeat sequences was generated by performing analignment analysis of five Alu consensus sequences representing the twofamily branches J and S (GenBank Acc. No. U14567 and U14571-14574)(Claverie, J-M and Makalowski, W. Science, 371; 752 (1994)), using theClustal W algorithm. The three identified subfamilies of the Y familybranch have not been included in the alignment shown in FIG. 2 as therelative frequency of Alu elements belonging to the Y family is very low(presumably less than 0.5% Sherry et al., Genetics, 147: 1977-1982(1997). The consensus sequence, and its complement, that were determinedusing this approach are Seq. ID. No. 1 and Seq. Id No. 2. The rawalignment data output for Seq. ID No. 1 is also presented in FIG. 2.

From the consensus sequence data, the nucleobase sequence of numerouspotential probes was determined. Generally however, the nucleobasesequence design of the various probes was selected to, as completely aspossible when the probes were mixed together, blanket the upper andlower strand of the Alu consensus sequence, as illustrated in FIG. 1, inorder to disrupt as much as possible the hybridization between theindividual strands of the repeated (Alu) sequences of the genomicnucleic acid of a sample. Because disruption occurs if probe is bound toone strand, the position for hybridization of the various probes, whenmixed together, was chosen such that the probes hybridizing to one ofthe two strands were substantially offset as compared with the probeshybridizing to the other strand. In this way the entire hybrid sequencewas blanketed with as few probes as possible. This approach was taken tominimize the number of probes need; it however is not a limitation sinceadditional probes or more extensive blanketing of the randomlydistributed repeat sequence is acceptable. Moreover, it was believedthat it was preferable to blanket at least fifty percent, and morepreferably at least two thirds, of the linear double stranded moleculeto thereby prevent the rehybridization of the two strands of genomicnucleic acid. Additionally, the probe candidates were checked for probeself dimers, probe pair dimers, and probe hairpins to minimize thehybridization reactivity with the probes themselves (i.e. intramolecularinteractions) and with other probes in the mixture (e.g. intermolecularinteractions).

Using these design parameters, the nucleobase sequences were chosen forthe numerous probes that might, when mixed together, suppress thebinding of detectable nucleic acid probes to undesired target genomicnucleic acid. These sequences were used to prepare non-nucleotide (i.e.PNA oligomer) probes for further testing and analysis.

Example 2: Evaluation of PNA Oligomer Candidates

Preferred Procedure for Performing In-Situ Hybridization Using DirectlyLabeled PNAs:

Slides for in-situ hybridization using directly labeled PNAs wereprepared as described in the general information on preferred method forcytogenetic preparations as discussed above. For pre-treatment, theslide containing metaphase spreads and interphase nuclei was immersedshortly in TBS (Tris-buffered saline), followed by 3.7% formaldehyde inTBS for 2 min at RT, and twice in TBS for 5 min each. The slide wastreated with Proteinase K (DAKO S3020, DAKO A/S, Glostrup, Denmark)diluted 1:2,000 in TBS for 10 min at RT, and rinsed twice in TBS for 5min each, followed by dehydration in a cold ethanol series (70%, 85%,and 96%), 2 min each. The slide was then air-dried. For hybridizationwith the fluorescein labeled PNA probes, 10 μL hybridization buffer (70%formamide, 20 mM Tris pH 7.5, 10 mM NaCl, 10 mM phosphate buffer pH 7.5,0.02% Ficoll, 0.02% polyvinylpyrrolidon, and 0.02% BSA (bovine serumalbumin)) with 50 nM PNA probe was added to the pre-treated slide. An18-mm² coverslip was applied to cover the hybridization mixture. Theslide was denatured by incubation at 80° C. for 5 min and allowed tohybridize by incubation at RT for 30 min. The coverslip was removed inPBS (Phosphate-buffered saline) at RT. Excess of probe was removed bywashing in preheated PBS with 1% Tween 20 at 60° C. for 25 min. Finallythe slide was dehydrated in a cool ethanol series and air-dried atdescribe above. The slide was mounted in 10 μL anti-fade mounting medium(Vectashield H-1000, Vector Laboratories, Inc. Burlingame) supplementedwith 0.1 μg/mL 4,6-diamoni-2phenyl-indole (DAPI, Sigma Chemicals) andsealed with a coverslip. Slides were then analyzed using a microscopeequipped with a CCD digital camera.

Digital Imaging Microscopy:

Images reproduced in FIGS. 3-7 were obtained using a Leica fluorescentmicroscope equipped with a 100× immersion oil objective, a 10× ocular(total enlargement is 1,000 fold) and fluorescent filter cubes obtainedeither from Leica or Chroma (Chroma Technology Corp., Brattleboro, Vt.,US). Electronic digital images were made of the slide using aPhotometric Sensys CCD-camera and Leica QFISH software (Leica ImagingSystem Ltd, Cambridge, UK).

Experimental Design:

It is well established that the hybridization of labeled nucleic acid,containing Alu sequences, to metaphase spreads will produce a distinctand highly reproducible R-banding pattern (Kornberg and Rykowski, 1988;Baldini and Ward, 1991). These isolated nucleic acid fragments aretypically greater than 100 bp in length. Thus, it seemed reasonable toprepare labeled non-nucleotide probes having the various chosennucleobase sequences and then determine whether or not they wouldproduce similar R-banding patterns as a way to test the binding affinityof these probes to randomly distributed repeat sequences in genomicnucleic acid and thereby score the result for each probe. Particularlybecause it was a consensus sequence that was being used to produce thePNA oligomers, and not one of the actual naturally occurring Alu-repeatsequences, testing was believed to be necessary to determine which ofthe nucleobase sequences hybridized most strongly to the Alu-repeatsequences of genomic nucleic acid.

For this purpose, a fluorescein labeled PNA oligomer was synthesized foreach nucleobase sequence candidate (chosen candidates are listed inTable 2). The various PNA constructs were then evaluated in a PNA-FISHassay using the procedure discussed above. The most important of theparameters to be analyzed was the R-banding potential but the relativeintensity of the R-banding signals was also examined. In each of thecategories, the R-banding potential and signal intensity for each of thePNA oligomers was assigned a score from the minimum value of 0 to amaximum value of 6. To select for PNA oligomers that had the nucleobasesequences that were most suitable for use in probe mixture for blockingof the Alu repeats, all oligomers having an R-banding potential limitvalue of less than 3 where eliminated. The process was used to screen atotal of 55 unique PNA oligomers, of which the 12 nucleobase sequencesidentified in Table 3 were found to be preferred because of theirsuperior ability to bind to genomic nucleic acid.

TABLE 3 Seq. Id. Nucleobase R-band Signal No. Position^(a) SequenceScore Intensity 3  9-25 GGCCGGGCGCG 4 5 GTGGCT 4 26-43 GCTGGGATTAC 5 6AGGCGTG 5 49-64 GGGAGGCCGAG 5 5 GCGGG 6  82-102 GCCAGGCTGGT 4 4CTCGAACTCC 7 112-132 GAAACCCCGTC 3 3 TCTACTAAAA 8 145-160 GCCGGGCGTGG 54 TGGCG 9 163-180 TAGCTGGGATT 6 5 ACAGGCG 10 184-200 GGGAGGCTGAG 6 6GCAGGA 11 201-221 CCTCCCGGGTT 3 3 CAAGCGATTC 12 228-244 TTGCAGTGAGC 3 3CGAGAT 13 253-273 TGCACTCCAGC 4 4 CTGGGCGACA 292^(c) ^(b)TT(k)TTTTT(k) 45 TTTLysOLysOTTT (k)TTTTT(k)TT ^(a)Refers to the relative positions inthe Alu consensus sequence depicted in FIG. 1. ^(b)Triplex makerconstruct. k = D-Lysine; Lys = L-lysine; O = 8-amino-3,6-dioxaoctanoicacid. ^(c)Hybridizes to the variable polyA tail of the alu sequence(Ullu E., TIBS: 216-219 (June, 1982)Results:

With reference to Table 3, there is data for each identified PNAoligomer that passed the limit value. For each entry, the relativeposition of the Alu consensus sequence, depicted in FIG. 2, isidentified and the recorded R-banding value and Signal intensity score(average of 4-6 independent experiments) is reproduced. Representativeexamples of fluorescein labeled PNA oligomers that obtained a high scorein the R-banding evaluation approach (Seq. Id No. 4 and Seq. Id. No. 10)and constructs that only just passed the limit value (Seq. Id. No. 7 andSeq. Id. No. 12) are shown in FIGS. 3A-1, 3A-2, 3B-1 and 3B-2,respectively. These images demonstrate that vast differences inperformance exist for the various probes examined and thereforedemonstrate that testing is a suitable way to determine which sequenceswere most effective at hybridizing to the randomly distributed repeatsequences of genomic nucleic acid.

Example 3: Effect of Sequence Variation

Experimental Design:

The Alu elements are dominated by the J and S family branches (Britten,R. J., Proc. Natl. Acad. Sci. USA, 91: 6148-6150 (1994); Batzer M A etal., J. Mol. Evol. (1996)). Especially noteworthy is the Sx subfamily,whereas the Y family branch constitute less than 0.5% of the total humanAlu elements. In order to test the sensitivity by which the R-bandingapproach can detect the frequency of a specific sequence within the Aluelements, comparisons of almost identical PNA oligomer constructs wereperformed.

The constructs to be compared were directed towards identical positionswithin the Alu consensus sequence (FIG. 2) but the specific nucleobasesequence design was based on either the J and S family consensus or theY family consensus. The images presented in FIG. 4 (A-D) were obtainedusing PNA oligomers that are complementary to positions 82-102 and201-221 of the sequence shown in FIG. 2 (the PNA oligomer sequences arecomplimentary to the depicted consensus sequence). Within positions82-102 the J and S consensus differ from the Y consensus by a T→Atransversion at position 86 and a G→T transversion at position 96.Similarly, within position 201-221 a single point mutation (or singlenucleotide polymorphism) correspondingly consists of a T→G transversionat position 208. The nucleobase sequence of the various PNA oligomerprobes used in the experiments, as well as the respective R-band andSignal intensity scores of the examined oligomer constructs (FIG. 4),are presented in Table 4. R-band and Signal intensity scores representaverage values from 8-10 independent experiments.

TABLE 4 Relative pos. in Seq. FIGS. Id. Alu Oligo 2-1- Signal No.consensus sequence  2-3 R-band Intensity  6 J and GCCAGGC  82-102 4 5S family TGGTCTC GAACTCC 27 Y family GCCAGGA  82-102 2 2 TGGTCTC GATCTCC11 J and CCTCCCG 201-221 3 3 S family GGTTCAA GCGATTC 28 Y familyCCTCCCG 201-221 1 1 GGTTCAC GCGATTCResults:

As can be seen by analysis of Table 4 and FIGS. 4 (A-D), these smallchanges (a single point mutation in one probe set and a double mutationin the other probe set) in the nucleobase sequence of the PNA oligomerssignificantly affects the efficiency by which the R-bands are formed. Itis not known whether the R-band signals from hybridization experimentsinvolving the Seq. Id. No. 27 and Seq. Id. No. 28 constructs (Y familyconsensus) reflect a low frequency of the corresponding genomicsequences, or rather that Seq. Id. No. 27 and Seq. Id. No. 28 hybridizeto the consensus sequences of the J and S family with a reducedaffinity. Nevertheless, this data reinforces the position that actualtesting should be performed in order to determine which of all possiblenucleobase sequences will produce the most complete hybridization to therandomly distributed repeat sequences and thereby presumably produce themost efficient blocking probes. Moreover, the aforementioned procedureappears to be well suited to determining the best candidates for furthertesting in a representative assay wherein there is substantialinterfering cross reaction of a detectable probe to undesired sequence.

Example 4: Suppression of Undesired Signal Using a PNA Probe Mixture

Preparation of DNA Probe/Blocking Agent Mixture:

Fluorescence labeled COS or PAC based DNA probes were prepare made byculturing the COS or PAC containing E. coli and harvesting the human DNAfrom the cultures. The COS or PAC DNA was purified using Qiagen largeconstruct kit (Qiagen, Kebolab A/S, Copenhagen, Denmark). The cloneswere checked by restriction enzyme digestion. From each clone, the DNAwas labeled by conventional Nick translation using a monomer labeledwith either fluorescein, Cy3, or Rhodamine. For the HER2 experimentsillustrated in FIG. 5, the COSs were labeled with fluorescein; for theMLL experiments in FIG. 6, the two PAC clones flanking the breakpoint(van der Burg et al., Leukemia 13: 2107-2113 (1999)) were labeled indifferent colors, one with fluorescein the other with Cy3; for the HER2experiments on tissue sections (FIG. 7) the COS clones were labeled withRhodamine. In all experiments the labeled COS or PAC DNA probes weremixed with a blocking agent (PNA Oligomer Mixture or Cot-1 DNA) in DNAHybridization Buffer (45% formamide, 300 mM NaCl, 5 mM NaPO₄, 10%Dextran sulphate). Each DNA probe was present at a final concentrationof 2 ng/μL. When using the PNA Oligomer Mixture as a blocking agent,each PNA oligomer was present at a concentration of 5 μM in thehybridization buffer. With the Cot-1 DNA as blocking agent, a 100:1weight ratio of Cot-1 DNA:total probe DNA was used in the assay.

Preferred Procedure for Performing In-Situ Hybridization UsingUnlabelled PNAs as Blocking Agent:

Slides containing metaphase spreads and interphase nuclei werepre-treated as described above. The slide was immersed in TBS with 3.7%formaldehyde for 2 minutes at RT and PBS for 2 minutes at RT followed bydehydration in chilled (5° C.) ethanol series (70%, 85%, and 96%); 2minutes each. On each pre-treated slide, 10 μL DNA of HybridizationBuffer with labeled DNA probe and unlabeled blocking agent (PNA OligomerMixture or Cot-1 DNA) is added and a 18 mm² coverslip is applied tocover the hybridization mixture. The edges of the coverslip were sealedwith rubber cement and air-dried until the cement had set (around 5min). The slide was denatured by incubation at 80° C. for 5 min. Theslides were then hybridized O.N. at 37° C. (when Cot-1 DNA is used asblocking agent) or 45° C. (when PNA Oligomer Mixture is used as ablocking agent). After hybridization the coverslip was removed and theslide rinsed at RT in 0.1×SSC followed by wash for 2×10 minutes in0.1×SSC at 55° C. (when Cot-1 DNA is used as the blocking agent) or 60°C. (when the PNA Oligomer Mixture is used as a blocking agent). Finallythe slide was dehydrated in 70%, 85%, and 96% EtOH and air-dried asdescribe above. Each slide was mounted with 10 μL anti-fade solution,with 0.1 μg/mL 4,6-diamoni-2phenyl-indole (DAPI, Sigma Chemicals) andsealed with a coverslip. Slides were then analyzed using a microscopeequipped with a CCD digital camera.

Experimental Design:

The Alu-banding approach (above) facilitated the identification ofnucleobase sequences, within the Alu repeats, that seem to be present inthe human genome with a high frequency. If Alu repeats are a majorreason for non-target hybridization of large probes of genomic origin, amixture of the identified PNA oligomer constructs should be able tosuppress this undesired hybridization background. Therefore, unlabelledPNA oligomers having the preferred nucleobase sequences, as identifiedin Table 3, were prepared. These unlabeled and purified probes weremixed together and used in hybridization experiments as previouslydescribed (the “PNA Oligomer Mixture”).

In addition to the unlabeled PNA probes, detectable nucleic acid probesof genomic origin were used in an assay to detect a genomic nucleic acidtarget. The detectable nucleic acid probes are COS clones coveringaround 100 kb of a region which include the HER-2 gene (17q21.1), or PACclones covering 90 kb on each side of MLL gene (11q23) major breakpointregion (mbr). For this experiment, the detectable nucleic acid probeswere labeled with fluorescein and the PNA oligomers were unlabeled.

In one experiment, the fluorescein labeled HER-2 probes were hybridizedto metaphase spreads and interphase nuclei. A representative microscopeimage of the resulting sample can be seen in FIG. 5A. This result wascompared with the standard art recognized blocking reagent, Cot1 DNA(FIG. 5B: Human Cot-1 DNA from Gibco BRL, Life Technologies). In aseparate sample, neither the PNA probe mixture nor the Cot1 DNA wasadded (FIG. 5C).

Translocations can be detected by a two color staining either by the“fusion” signal principle or the “split” signal principle. In the fusionsignal principle the two genes involved in the translocation are labeledin each a separate color. In cells where a translocation has taken placethe two colors will come together as a “fusion” signal. In the “split”signal principle the two differently labeled probes are localized aroundthe breakpoint in one of the genes participating in the translocation(van der Burg et al., Leukemia 13: 2107-2113 (1999)). Thus, in abnormalcells the two probes will split as a result of the translocation.Similarly, the MLL probes labeled with fluorescein or Cy3 werehybridized to metaphase spreads and interphase nuclei using the sameprocedure as used for the HER 2 probes. A representative microscopeimage of the resulting sample can be seen in FIG. 6A. This result wascompared with the standard art recognized blocking reagent, Cot1 DNA(FIG. 6B). In a separate sample, neither the PNA probe mixture nor theCot1 DNA was added (FIG. 6C).

With reference to FIGS. 5 A-C and 6 A-C, it is apparent that in bothcases the mixture of PNA probes appears to work as well as, if notbetter than, the industry standard, Cot1 DNA. It is also noteworthy thatthe absence of any blocking agent (e.g. PNA probe mixture or the Cot1DNA) results in the formation of a hybridization background with aR-banding pattern, thereby indicating that non-target hybridization iscaused by the presence of Alu repeats or other randomly distributedrepeat sequence. The intensity of this background also seriously hindersthe visualization of the single locus specific signals such that it mustbe removed in order to facilitate the performance of an accurate andreproducible assay.

Example 5: Suppression of Undesired Background in Tissue Sections Usingthe PNA Probe Mixture

The PNA blocking mixture was used to suppress the undesired backgroundstaining from the HER2 probes described in Example 4 when used on tissuesections from a breast carcinoma. The DNA probes were labeled withRhodamine and mixed with either the PNA Oligomer Mixture (FIG. 7A) orCot-1 DNA (FIG. 7B). For comparison FIG. 7C shows the same experimentwithout any blocking agent added.

Preferred Procedure for Performing In-Situ Hybridization to ParaffinEmbedded Mamma Carcinoma Sections Using Unlabelled PNAs as BlockingAgent:

Mamma carcinoma sections of 4 μm were cut from paraffin embedded tissueblocks. Slides mounted with tissue sections were deparaffinated inXylene and 96% EtOH according to standard procedures (DAKO's handbook:Immunochemical Staining Methods). The slides were pre-treated in PBS for10 min. at RT and 10 min. in boiling MES(2-[N-morpholino]ethanesulphonic acid) buffer, pH 6.4 followed bydigestion in a 0.05% pepsin solution (0.05% pepsin in 0.02M HCl, 0.9%NaCl) for 10 min. at 37° C. The slides are washed for 3×2 minutes in PBSand dehydrated in chilled (5° C.) ethanol series (70%, 85%, and 96%EtOH, 2 minutes each). The slides were allowed to air-dry. Forhybridization, 10 μL DNA hybridization buffer containing the labeled DNAprobe and a blocking agent was added to the pre-treated slide and a 18mm² coverslip was applied to cover the hybridization mixture. The edgesof the coverslip were sealed with rubber cement and air-dried until thecement had set (around 5 min). The slide was denatured by incubation at90° C. for 5 min. The slides were hybridized O.N. at 45° C. Afterhybridization the coverslip was removed and the slide was rinsed inpre-warmed (55° C.) 0.2×SSC with 0.1% TritonX-100 followed by a wash for10 minutes in 0.2×SSC with 0.1% TritonX-100 at 55° C. Finally the slidewas rinsed in PBS for 2 minutes at RT followed by dehydration in chilledethanol series (70%, 85%, and 96% EtOH, 2 minutes each) and air-dried asdescribed above. Each slide was mounted with 10 μL anti-fade solutionwith 0.1 μg/mL 4,6-diamoni-2phenyl-indole (DAPI, Sigma Chemicals) andsealed with a coverslip. Slides were then analyzed using a microscopeequipped with a CCD digital camera.

Results:

With reference to FIGS. 7A and 7B it is clear that there is very littledifference in the performance of the PNA probe mixture vs. the Cot-1DNA. Moreover, the absence of any blocking agent results in substantialbackground (FIG. 7C).

Example 6: More Examples of Chromosome Analysis Synthesis and Labelingof Peptide Nucleic Acid Probes

The chromosome 17 centromere Peptide Nucleic Acids (PNAs) weresynthesized at the 2-μmole scale on an Expedite 8900 nucleic acidsynthesis system (Applied Biosystems, Foster City, Calif.) using Fmocchemistry. The Alu PNAs were synthesized at the 5 μmole scale using a433A nucleic acid synthesis system (Applied Biosystems, Foster City,Calif.) using t-boc chemistry. All of the PNAs were solubility enhancedusing compound 4 as described (Gildea et al 1998). Attachment of alinker group while the oligomer was still bound to the column wasaccomplished by condensation of the expedite PNA linker,Fmoc-8-amino-3,6-dioxaotanoic acid. For 5(6)-carboxyfluorescein labelingthe synthesis support was heated at 30° C. for five hours in 250 μL of asolution containing 0.08M dye as an NHS ester, (Molecular Probes,Eugene, Oreg.), 0.25M diisopropylethylamine and 0.2-M lutidine. Thecrude oligomer samples were then cleaved from the support by the use ofstandard methods, precipitated and purified by high performance liquidchromatography (HPLC) using 0.1% trifluoroacetic acid and a linearacetonitrile gradient.

Preparation of Chromosome Specific Probes

Chromosome 17 specific PNA probes (18-22 base units) were selected frompublished data available in public databases (e.g. Genbank). Thefollowing seven “Chromosome 17 PNA Probes” specific for the chromosome17 α-satellite sequences were selected;

1. Flu-OEE-AAC-GAA-TTA-TGG-TCA-CAT-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 55)2. Flu-OEE-GGT-GAC-GAC-TGA-GTT-TAA-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 56)3. Flu-OEE-AAC-GGG-ATA-ACT-GCA-CCT-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 57)4. Flu-OEE-ATC-ACG-AAG-AAG-GTT-CTG-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 58)5. Flu-OEE-TTT-GGA-CCA-CTC-TGT-GGC-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 59)6. Flu-OEE-GAA-TCT-TCA-CAG-GAA-AGC-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 60)7. Flu-OEE-GAT-TCT-ACA-CAA-AGA-GAG-EEO-Lys(Flu)-NH₂ (SEQ ID NO: 61)(Abbreviations are as previously described)

GenBank accession numbers U14567, U14568, U14569, U14570, U14571,U14572, U14573, U14574 representing the Alu consensus sequences wereused to select sequences specific for the Alu family of interspersedrepeats. The following eleven “Alu PNA Blocking Probes” selected fromthe consensus sequences were as follows:

 1. H-EE-TTG-CAG-TGA-GCC-GAG-AT-EE-NH₂ (SEQ ID NO: 62) 2. H-EE-GGC—CGG-GCG-CGG-TGG-CT-EE-NH₂ (SEQ ID NO: 63) 3. H-EE-GCT-GGG-ATT-ACA-GGC-GTG-EE-NH₂ (SEQ ID NO: 64) 4. H-EE-GGG-AGG-CCG-AGG-CGG-G-EE-NH₂ (SEQ ID NO: 65) 5. H-EE-GCC-AGG-CTG-GTC-TCG-AAC-TCC-EE-NH₂ (SEQ ID NO: 66) 6. H-EE-GAA-ACC—CCG-TCT-CTA-CTA-AAA-EE-NH₂ (SEQ ID NO: 67) 7. H-EE-GCC-GGG-CGT-GGT-GGC-G-EE-NH₂ (SEQ ID NO: 68) 8. H-EE-TAG-CTG-GGA-TTA-CAG-GCG-EE-NH₂ (SEQ ID NO: 69) 9. H-EE-GGG-AGG-CTG-AGG-CAG-GA-EE-NH₂ (SEQ ID NO: 70)10. H-EE-CCT-CCC-GGG-TTC-AAG-CGA-TTC-EE-NH₂ (SEQ ID NO: 71)11. H-EE-TGC-ACT-CCA-GCC-TGG-GCG-ACA-EE-NH₂ (SEQ ID NO: 72)(Abbreviations are as previously described)In-Situ Hybridization

(i) Slide Preparation

Slides containing metaphase spreads were prepared with standardcytogenetic techniques, essentially as previously described herein, andaged overnight then stored at −20° C. Prior to hybridization, the slideswere removed from the freezer and allowed to warm to room temperature.

(ii) Hybridization and Washings

To 10 μL of hybridization mix containing 45% formamide, 10% dextransulphate, 300 mM NaCl, 5 mM Na phosphate, 100 ng of rhodamine labeledHer2 DNA probe, and optionally as discussed below either or both of: (a)the Chromosome 17 PNA Probes at 30 nM each and/or (b) Alu PNA BlockingProbes at 5 μM each, were added to the slide. The slides were denaturedat 70° C. for six minutes followed by hybridization overnight at 37° C.in a humidified chamber. After removal of the coverslip the slides arewashed in a stringent wash solution (0.2×SSC with 0.1% Triton X-100) at65° C. for 10 minutes. The slides were then rinsed in TBS buffer for 2minutes. Cells were counterstained with DAPI and mounted in Vectashieldanti-fade medium.

(iii) Digital Imaging Microscopy

Digital images of metaphase cells after FISH with fluorescein labeledChromosome 17 PNA Probes and/or Rhodamine labeled Her2 DNA probes wereacquired with a cool snap FX 12 bit CCD camera (Roper Scientific, TucsonAriz.) attached to an Olympus AX 70 microscope using Openlab software(Improvision Inc., Lexington, Mass.). The microscope was equipped withSP100, 41001, SP102, SP102, SP104 and SP105 filter sets for multicolorFISH (Chroma Technology, Brattleboro, Vt.). Images of each fluorescentdye were acquired, avoiding over or under exposure and stored forfurther analysis. After thresholding and contrast enhancement usingOpenlab software (Improvision Inc., Lexington, Mass.), pixels above aselected threshold from each fluorescent dye were projected on to theDAPI image.

Results:

As described above, a series of experiments were designed to establishthe usefulness of a mixture of Alu PNA Blocking Probes in the analysisof genomic nucleic acid in FISH experiments. First, the affect ofhybridizing a rhodamine labeled genomic DNA probe (rhodamine labeledHer2 DNA) in the absence of Alu PNA Blocking Probes was used as acontrol. With respect to FIG. 8A, the interphase and metaphase cellswere stained bright red with non-specific staining clearly visible.There was little or no differentiation between the specific Her2 signaland the non-specific background staining. In addition, the morphology ofthe chromosomes was extremely poor.

By comparison, the affect of hybridizing the same labeled genomic probe(rhodamine labeled Her2 DNA), wherein Alu PNA Blocking Probes werepresent, was performed. With respect to FIG. 8B, the interphase andmetaphase cells contain low non-specific staining and bright specificred Her2 signal on the long arm of chromosome 17. This demonstrates thatthe presence of the Alu PNA Blocking Probes significantly reducenon-specific hybridization.

To confirm that the observed signal was present on chromosome 17, andnot on another chromosome, in the next series of experiments a mixtureof Fluorescein Chromosome 17 PNA Probes were included in thehybridization mix along with the rhodamine labeled Her2 DNA probe, bothwith (FIG. 8C) and without Alu PNA Blocking Probes (FIG. 8D).

With reference to FIG. 8C, hybridization of the rhodamine labeled Her2DNA probe and the Fluorescein Chromosome 17 PNA Probe mix, in theabsence of Alu PNA Blocking Probes, produces results that are similar toFIG. 8A, wherein the Fluorescein Chromosome 17 PNA Probe are omittedexcept there was non-specific binding of the probe to the glass slide.Specifically, there is too much non-specific signal for the assay to beof practical utility. By comparison, FIG. 8D shows the effect ofincluding the Alu blocking probes. In this Figure there is a bright redspecific Her2 signal on the long arm of chromosome 17 (as indicated bythe arrows) with low non-specific staining of the interphase cells andmetaphase chromosomes with little or no non specific hybridization onthe glass slide. Because the Fluorescein Chromosome 17 PNA Probes werepresent in the mix, it is confirmed that the green signal correlateswith the red signal, thereby confirming that the DNA probe hybridizesspecifically to chromosome 17 and not another chromosome.

Example 7: Detection of Translocation of the CyclinD1 Gene UsingSuppression of Undesired Background with the PNA Oligomer Mixture

Preparation of DNA Probe/Blocking Agent Mixture:

Fluorescence labeled BAC based DNA probe was prepare made by culturingthe BAC containing E. coli and harvesting the human DNA from thecultures. The BAC DNA was purified using Qiagen large construct kit(Qiagen, Kebolab A/S, Copenhagen, Denmark). The clone was checked byrestriction enzyme digestion and end sequenced. The clone DNA waslabeled by conventional Nick translation using a monomer labeled withfluorescein. In all experiments the labeled DNA probe was mixed with ablocking agent (with or without PNA Oligomer Mixture or Cot-1 DNA) inDNA Hybridization Buffer (45% formamide, 300 mM NaCl, 5 mM NaPO₄, 10%Dextran sulphate). The DNA probe was present at a final concentration of5 ng/μL. The centromeric PNA probe was labeled with Rhodamine andpresent at a final concentration of 50 nM. When using the PNA OligomerMixture as a blocking agent, each PNA oligomer was present at aconcentration of 5 μM in the Hybridization Buffer. With the Cot-1 DNA asblocking agent, a 100:1 weight ratio of Cot-1 DNA:total probe DNA wasused in the assay.

Preferred Procedure for Performing In-Situ Hybridization UsingUnlabelled PNAs as Blocking Agent:

Slides containing metaphase spreads and interphase nuclei werepre-treated as described above. The slides were immersed in TBS with3.7% formaldehyde for 2 minutes at RT and PBS for 2 minutes at RTfollowed by dehydration in chilled (5° C.) ethanol series (70%, 85%, and96%); 2 minutes each. On each pre-treated slide, 10 μL DNA ofHybridization Buffer with labeled DNA probe and unlabeled blocking agent(PNA Oligomer Mixture or Cot-1 DNA) is added and a 18 mm² coverslip isapplied to cover the hybridization mixture. The edges of the coverslipwere sealed with rubber cement before treatment at 80° C. for 5 min. Theslides were then hybridized O.N. at 45° C. After hybridization, thecoverslip was removed and the slide rinsed at RT in Stringent WashBuffer (0.2×SSC, 0.1% Triton X-100) followed by wash for 10 minutes inStringent Wash Buffer at 65° C. Finally the slide was dehydrated in 70%,85%, and 96% EtOH and air-dried as describe above. Each slide wasmounted with 10 μL anti-fade solution, with 0.1 μg/mL4,6-diamoni-2-phenyl-indole (DAPI, Sigma Chemicals) and sealed with acoverslip. Slides were then analyzed using a microscope equipped with aCCD digital camera.

Experimental Design:

The Alu-banding approach (above) facilitated the identification ofnucleobase sequences, within the Alu repeats, that seem to be present inthe human genome with a high frequency. If Alu repeats are a majorreason for non-target hybridization of large probes of genomic origin, amixture of the identified PNA oligomer constructs should be able tosuppress this undesired hybridization background. Therefore, unlabelledPNA oligomers having the preferred nucleobase sequences, as identifiedin Table 3, were prepared. These unlabeled and purified probes weremixed together and used in hybridization experiments as previouslydescribed.

Results:

Since the CyclinD1 gene is located on chromosome band 11q23, a PNAmixture for the centromere of chromosome 11 (See U.S. Ser. No.09/520,760 or U.S. Ser. No. 09/627,796, herein incorporated byreference) was prepared and added to the assay to thereby identify thechromosome to which the CyclinD1 probe hybridizes. The PNA oligomers forthe centromere of chromosome 11 were labeled with Rhodamine. FIG. 9Ashows both centromeres of chromosome 11 in red, as well as the greenCyclinD1 probes. FIG. 9B shows the same probes wherein Cot-1 DNA is usedas compared with the PNA Oligomer Mixture. The red centromere signalsare not visible as Cot-1 also contains centromeric sequences and thuscompete with the PNA in binding to the centromere. FIG. 9C show the sameprobes without any blocking agent added. It is not possible to identifyeither of the red or the green signals in the interphase nuclei. In themetaphase spread, only the centromere signals (in red) can be seen.

The CyclinD1 probe and the Centromere 11 PNA was also hybridized tometaphase spreads of the cell line Granta 519 ((Drexler, H. G. (2001)The Leukemia-Lymphoma Cell Line Facts Book. Braunschweig, Germany)) thatharbors a translocation of the CyclinD1 gene. The probe was labeled withfluorescein and mixed with either the PNA Oligomer Mixture (FIG. 9D) orCot-1 DNA (FIG. 9E). For comparison FIG. 9C shows the same experimentwithout any blocking agent added.

Example 8: Detection of Amplification of the c-MYC Gene UsingSuppression of Undesired Background with the PNA Oligomer Mixture

The c-MYC DNA probe was prepared as described in example 7. Furthermore,a DNA based centromere probe was used instead of a PNA based centromereprobe. The DNA centromere DNA probe was prepare by culturing thecentromere DNA plasmid containing E. coli and harvesting the human DNAfrom the cultures. The plasmid DNA was purified using Qiagen plasmid kit(Qiagen, Kebolab A/S, Copenhagen, Denmark). The clone DNA was labeled byconventional Nick translation using a monomer labeled with fluorescein.The DNA based centromere probe was used at a final concentration of 2pg/μL. The procedure for performing in-situ hybridization is describedin Example 7.

Results:

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from a c-MYC probe when used on normal metaphase spreads andinterphase nuclei. The c-MYC probe was labeled with Texas Red and mixedwith either the PNA Oligomer Mixture (FIG. 10A) or Cot-1 DNA (FIG. 10B).For comparison FIG. 10C shows the same experiment without any blockingagent added. Since the c-MYC gene is located on chromosome band 8q24, aDNA probe for the centromere of chromosome 8 was added to identify thechromosome to which the c-MYC probe hybridizes (See discussion abovewith respect to the preparation of the DNA centromere probe forchromosome 8). The DNA for centromere 8 was labeled with fluorescein.FIG. 10A shows both centromeres clearly and both genes localized onchromosome 8. In the same Figure an interphase nuclei shows two separatered and two separate green signals thereby confirming the determinationof the c-MYC gene on chromosome 8. FIG. 10B shows the result when usingCot-1 DNA instead of the PNA Oligomer Mixture. The green centromeresignals are not visible as Cot-1 also contains centromeric sequences andthus compete with the DNA in binding to the centromere. FIG. 10C showthe same experiment without any blocking agent added (i.e. no PNAOligomer Mixture and no Cot1-PNA). Only the centromere signals (ingreen) can be clearly seen. The red signals from the cMYC gene can notbe seen due to the high background staining.

The c-MYC probe and the centromere 8 DNA probe were also hybridized tometaphase spreads of the cell line HMT3522 (Nielsen et al., 1997) thathas amplification of the c-MYC gene. The c-MYC probe was labeled withTexas Red, the DNA probe for the centromere of chromosome 8 was labeledwith fluorescein. These probes were mixed with either the PNA OligomerMixture (FIG. 10D) or Cot-1 DNA (FIG. 10E). For comparison, FIG. 10Fshows the result of the same experiment performed without any blockingagent added. Using the PNA Oligomer Mixture both the green centromere 8signals and the amplified c-MYC signals (in red) can be seen. UsingCot-1 DNA only the red c-MYC signals can bee seen. Omitting the blockingagent makes it impossible to discriminate between red signals andbackground.

Ref: Nielsen K V, Niebuhr E, Ejlertsen 13, Holstebroe S, Madsen M W,Briand P, Mouridsen H T, Bolund L. Molecular cytogenetic analysis of anontumorigenic human breast epithelial cell line that eventually turnstumorigenic: Validation of an analytical approach combining karyotyping,comparative genomic hybridization, chromosome painting, and single-locusfluorescence in situ hybridization. Genes Chromosomes Cancer. 1997;20:30-37.

Example 9: Detection of Amplification of the EGFR Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA blocking mixture was used to suppress the undesired backgroundstaining from a EGFR probe when used on normal metaphase spreads andinterphase nuclei. The EGFR probe was prepared essentially as describedin Example 8. The procedure for in-situ hybridization on tissue wasperformed essentially as described in Example 5. The procedure forin-situ hybridization on metaphase spreads was performed essentiallydescribed in Example 7.

Results:

The EGFR gene is located on the short arm of chromosome 7. Therefore aDNA probe for the centromere of chromosome 7 was added to identify thechromosome to which the EGFR probe hybridizes. The DNA for centromere 7was labeled with fluorescein. The EGFR probe was labeled with Texas Redand mixed with either the PNA Oligomer Mixture (FIG. 11A) or Cot-1 DNA(FIG. 11B). For comparison FIG. 11C shows the result of the sameexperiment performed without any blocking agent added.

FIG. 11A shows both centromeres clearly and both genes localized onchromosome 7. FIG. 11B shows the same probes except that Cot-1 DNA wassubstituted for the PNA Oligomer Mixture. The green centromere signalsare nearly invisible as Cot-1 also contains centromeric sequences andthus compete with the DNA in binding to the centromere. FIG. 11C showsthe result of performing the assay in the absence of any blocking agent.Only the centromere signals (in green) can be seen clearly. The redsignals from the EGFR gene are difficult to distinguish from the highbackground staining.

The EGFR probe and the centromere 7 DNA probe were also hybridized tohuman lung tissue that has amplification of the EGFR gene. The EGFRprobe and the DNA probe for the centromere of chromosome 7 were mixedwith either the PNA Oligomer Mixture (FIG. 11D) or Cot-1 DNA (FIG. 11E).For comparison, FIG. 11F shows the result of the same experimentperformed without any blocking agent added. Using the PNA OligomerMixture both the green centromere 7 signals and the amplified EGFRsignals (in red) can be seen. Using Cot-1 DNA only the red EGFR signalscan bee seen. Omitting blocking agent interferes marginally with signalrecognition, since only a slight increase in background staining isseen.

Example 10: Suppression of Undesired Background Using the PNA OligomerMixture in Combination with a TOP2A DNA Probe and a CEN-17 PNA Probe

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from the TOP2A DNA probe and CEN-17 PNA probe. This assay hasbeen launched as a kit by DakoCytomation with the product code no.K5333. The TOP2A DNA probe, labeled with Texas Red and the CEN-17 PNAprobe labeled with fluorescein were prepared essentially as described inExample 4. A mixture of the PNA and DNA probes was tested on normalmetaphase spreads (FIGS. 12A, 12B and 12C), essentially as described inExample 7 and on formalin fixed, paraffin embedded cells from the cellline MDA-361 (breast cancer cell line with TOP2A deletion (Jarvinen T A,Tanner M, Rantanen V, Barlund M, Borg A, Grenman S, et al. Amplificationand deletion of topoisomerase IIalpha associate with ErbB-2amplification and affect sensitivity to topoisomerase II inhibitordoxorubicin in breast cancer. Am J Pathol 2000; 156: 839-4; FIGS. 12D,12E and 12F) and a mama carcinoma with borderline amplification (FIGS.12G, 12H and 12I) essentially as described in Example 5. The TOP2A DNAprobe and CEN-17 PNA probe (See U.S. Ser. No. 09/520,760 or U.S. Ser.No. 09/627,796, herein incorporated by reference) were mixed with eitherthe PNA Oligomer Mixture (FIGS. 12A, 12D and 12G) or Cot-1 DNA (FIGS.12B, 12E and 12H). For comparison FIGS. 12C, 12F and 12I show the resultof performing the same assay in the absence of any blocking agent.

Results:

FIGS. 12A, 12B and 12C demonstrates the ability for the PNA OligomerMixture to block unspecific background when detecting the TOP2A gene onnormal metaphase spreads using the green CEN-17 PNA probe as areference. Green and red signal are clearly visible using the PNAmixture as a blocking reagent (12A), whereas the green signals aresomewhat dimmer when using Cot-1 DNA as a blocking reagent (12B).Specific signals cannot be distinguished from background in the absenceof any blocking reagent (12C).

FIGS. 12D, 12E and 12F demonstrates the ability of the PNA OligomerMixture to block unspecific background when detecting a TOP2A genedeletion in MDA-361 cells, using CEN-17 as a reference. Green and redsignal are clearly visible using either the PNA Oligomer Mixture (12D)or Cot-1 DNA as a blocking reagent (12E). Specific signals cannot bedistinguished from background in the absence of any blocking reagent(12F).

FIGS. 12G, 12H and 12I demonstrate the ability for the PNA OligomerMixture to block unspecific background when determining the ratiobetween the TOP2A gene and CEN-17 in a human mama carcinoma. Green andred signal are clearly visible using either the PNA Oligomer Mixture(12G) or Cot-1 DNA (12H) as a blocking reagent. Specific signals cannotbe distinguished from background in the absence of any blocking reagent(12I).

Example 11: Detection of Translocation of the TEL Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from TEL probes when used on normal metaphase spreads andinterphase nuclei. The TEL probes was prepared essentially as describedin Example 7, provided however that BAC DNA and PAC DNA were used. Theupstream clone DNA was labeled with Texas Red and the downstream cloneDNA was labeled with fluorescein by conventional Nick translation usinga monomer labeled with Texas Red or fluorescein, as appropriate. The DNAbased probes were used at a final concentration of 50 ng/μL. Theprocedure for performing in-situ hybridization is essentially asdescribed in Example 7. The DNA probes were mixed with either the PNAOligomer Mixture (FIG. 13A) or Cot-1 DNA (FIG. 13B). For comparison FIG.13C shows the same experiment without any blocking agent added.

The TEL gene is located on chromosome band 12p13. FIG. 13A shows normalTEL configurations on metaphases with a yellow signal located on bothchromosome 12. Two yellow signals in interphase nuclei indicate twonormal TEL loci. FIG. 13B shows the result using Cot-1 DNA instead ofthe PNA Oligomer Mixture. FIG. 13C shows the result of the same assayexcept that no blocking agent added. It is not possible to identifyeither the red or the green signals in the interphase nuclei andmetaphase spread in the absence of a blocking reagent.

The TEL probes were also hybridized to metaphase spreads of the cellline REH (Drexler, H. G. (2001) The Leukemia-Lymphoma Cell Line FactsBook. Braunschweig, Germany.) that harbors a translocationt(12;21)(p13,q22). The assay was performed with either the PNA OligomerMixture (FIG. 13D) or Cot-1 DNA (FIG. 13E). FIG. 13D and FIG. 13E showsone green signals located on der(12) and a red signal on der(21),indicating a split of the upstream TEL and downstream TEL probes. Thisis indicative of a translocation. A green signal on the other allele ofchromosome 12 would indicate a deletion of the upstream TEL. Forcomparison, FIG. 13F shows the result of performing the same assay inthe absence of any blocking agent.

Example 12: Detection of Translocation of the E2A Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from E2A probes when used on normal metaphase spreads andinterphase nuclei. The BCR probes was prepared essentially as describedin Example 11. In-situ hybridization was performed essentially asdescribed in Example 7.

The upstream E2A probe was labeled with fluorescein and the downstreamE2A probe with Texas Red. The E2A gene is located on chromosome band19p13. FIG. 14A show normal E2A configurations on metaphases with ayellow signal (mixture of red and green signals) located on bothchromosome 19. Two yellow signals in interphase nuclei indicate twonormal E2A loci. FIG. 14B show the result when the assay is performedwith Cot-1 DNA instead of the PNA Oligomer Mixture. FIG. 14C show theresult when the assay is performed without any blocking agent. It is notpossible to identify neither the red nor the green signals in theinterphase nuclei and metaphase spread.

The E2A probes were also hybridized to metaphase spreads of the cellline 697 (Drexler, H. G. (2001) The Leukemia-Lymphoma Cell Line FactsBook. Braunschweig, Germany) that harbors a translocationt(1;19)(q23,p13). The E2ADNA probes were labeled as above and mixed witheither the PNA Oligomer Mixture (FIG. 14D) or Cot-1 DNA (FIG. 14E). FIG.14D and FIG. 14E shows one yellow signal indicating a normal E2A alleleand a red signal on der(1) while the der(19) is lost, indicating a splitof the upstream BCR and downstream BCR probes. This result is indicativeof a translocation. For comparison FIG. 14F shows the result ofperforming the assay in the absence of any blocking agent.

Example 13: Detection of Translocation of the BCR Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from BCR probes in normal metaphase spreads and interphasenuclei. The upstream BCR DNA probe was labeled with fluorescein and thedownstream BCR DNA probe with Texas Red. The BCR probes were preparedessentially as described in Example 11. However, COS DNA was alsoincluded as probe. The COS DNA was labeled with Texas Red byconventional Nick translation using a monomer labeled with Texas Red.The procedure for performing in-situ hybridization was performedessentially as described in Example 7.

These DNA probe were mixed with either the PNA Oligomer Mixture (FIG.15A) or Cot-1 DNA (FIG. 15B). For comparison FIG. 15C shows the resultof performing the same assay in the absence of blocking agent.

The BCR gene is located on chromosome band 22q11. FIG. 15A show normalBCR configurations on metaphases with a yellow signal located on bothchromosome 22. Two yellow signals in interphase nuclei indicate twonormal BCR loci. FIG. 15B show the same probes suppressed with Cot-1DNA. FIG. 15C show the same probes without any blocking agent added. Itis not possible to identify the red or the green signals in theinterphase nuclei and metaphase spread in the absence of a blockingreagent.

The BCR probes were also hybridized to metaphase spreads of the cellline BV173 (Drexler, H. G. (2001) The Leukemia-Lymphoma Cell Line FactsBook. Braunschweig, Germany) that harbors a translocationt(9;22)(q34,q11). The BCR probes were labeled as above and mixed witheither the PNA Oligomer Mixture (FIG. 15D) or Cot-1 DNA (FIG. 15E). FIG.15D and FIG. 15E shows one yellow signal, indicating a normal BCRallele, and one green signals located der(9) and a red signal onder(22); indicating a split of the upstream BCR and downstream BCRprobes. This result is indicative of a translocation. An additional redsignal is indicative of an additional der(22). For comparison, FIG. 15Fshows the result of performing the same assay in the absence of blockingagent.

Example 14: Detection of Translocation of the IGH Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from IGH probes when used on normal metaphase spreads andinterphase nuclei. The probes were labeled with fluorescein for the IGHVgenes and with Texas Red for the IGHC genes (Poulsen T S, Silahtaroglu AN, Gisselo C G, Gaarsdal E, Rasmussen T, Tommerup N, Johnsen H E.Detection of illegitimate rearrangement within the immunoglobulin locuson 14q32.3 in B-cell malignancies using end-sequenced probes. (2001)Genes Chromosomes Cancer, 32: 265-74), mixed with either the PNAOligomer Mixture (FIG. 16A) or Cot-1 DNA (FIG. 16B). The IGH probes wereprepared as described in Example 11. In-situ hybridization was performedessentially as described in Example 11. However, in this Example 14, thepre-treatment was omitted and hybridization time was lowered to 4 hours,thus showing the robustness of the assay.

The DNA probes were mixed with either the PNA Oligomer Mixture (FIG.16A) or Cot-1 DNA (FIG. 16B). For comparison FIG. 16C shows the resultof performing the same assay in the absence of blocking agent.

The IGH genes are located on chromosome band 14q32. FIG. 16A showsnormal IGH configurations on metaphases with a yellow signal located onboth chromosome 14, band q32. FIG. 16B shows the result when backgroundis suppressed using Cot-1 DNA. FIG. 16C shows the result of performingthe assay in the absence of a blocking agent. It is not possible toidentify the red or the green signals in the interphase nuclei andmetaphase spread in the absence of a blocking agent.

The IGH probes were also hybridized to metaphase spreads of the cellline Granta 519 (Drexler, H. G. (2001) The Leukemia-Lymphoma Cell LineFacts Book. Braunschweig, Germany) that harbors a translocationt(11;14)(q23,q32). The IGH probes were labeled as above and mixed witheither the PNA Oligomer Mixture (FIG. 16D) or Cot-1 DNA (FIG. 16E). FIG.16D and FIG. 16E shows one yellow signal located on 14q32, indicating anormal IGH configuration on one allele and a split of the IGHV and IGHCprobes on the other allele. This result is indicative of atranslocation. For comparison FIG. 16F shows the same experiment withoutany blocking agent added.

Example 15: Detection of Translocation of the IGL Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from IGL probes when used on normal metaphase spreads andinterphase nuclei. The probes were labeled with fluorescein for the IGLVgenes and with Texas Red for the IGLC genes (Poulsen T S, Silahtaroglu AN, Gisselo C G, Gaarsdal E, Tommerup N, Johnsen H E. Detection ofillegitimate rearrangements within the immunoglobulin light chain lociin B-cell malignancies using end sequenced probes. (2002) Leukemia, 16:2148-2158), mixed with either the PNA Oligomer Mixture (FIG. 17A) orCot-1 DNA (FIG. 17B). The IGL probes were prepared essentially asdescribed in Example 11. The procedure for performing in-situhybridization was performed essentially as described in Example 11.However, the hybridization time was lowered to 4 hours, thus showing therobustness of the assay.

The DNA probes were mixed with either the PNA Oligomer Mixture (FIG.17A) or Cot-1 DNA (FIG. 17B). For comparison FIG. 17C shows the resultof performing the same assay in the absence of blocking agent.

The IGL genes are located on chromosome band 22q11. FIG. 17A show normalIGL configurations on metaphases with a yellow signal located on bothchromosome 22, band q11. FIG. 17B shows the result of the assay whenCot-1 DNA is used. FIG. 17C shows the result of performing the assay inthe absence of a blocking agent. It is not possible to identify the redor the green signals in the interphase nuclei and metaphase spread inthe absence of a blocking agent.

Example 16: Detection of Translocation of the IGK Gene Using Suppressionof Undesired Background with the PNA Oligomer Mixture

The PNA Oligomer Mixture was used to suppress the undesired backgroundstaining from IGL probes when used on normal metaphase spreads andinterphase nuclei. The probes were labeled with fluorescein for the IGKVgenes and with Texas Red for the IGKC genes (Poulsen T S, Silahtaroglu AN, Gisselo C G, Gaarsdal E, Tommerup N, Johnsen H E. Detection ofillegitimate rearrangements within the immunoglobulin light chain lociin B-cell malignancies using end sequenced probes. (2002) Leukemia, 16:2148-2158). The IGK genes are located on chromosome band 2p11. The IGKDNA probes were prepared essentially as described in Example 11. In-situhybridization was performed essentially as described in Example 11,provided however, that the hybridization time was lowered to 4 hours,thus showing the robustness of the assay.

The DNA probes were mixed with either the PNA Oligomer Mixture (FIG.18A) or Cot-1 DNA (FIG. 18B). For comparison, FIG. 18C shows the resultof performing the assay in the absence of any blocking agent. FIG. 18Ashow normal IGK configurations on metaphases with a yellow signallocated on both chromosome 2, band p11. FIG. 18B show the same probessuppressed with Cot-1 DNA. FIG. 18C show the same probes without anyblocking agent added. It is not possible to identify neither the red northe green signals in the interphase nuclei and metaphase spread.

Summary of Experimental Section

The aforementioned Examples 1-16, when taken together, demonstrate theutility of both the method for production of the PNA Oligomer Mixture aswell as for use in suppressing the binding of detectable nucleic acidprobes to randomly distributed repeat sequence in genomic nucleic acid.

EQUIVALENTS

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose of ordinary skill in the art that various changes in form anddetails may be made therein without departing from the spirit and scopeof the invention as defined by the appended claims. Those skilled in theart will be able to ascertain, using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. Such equivalents are intended to beencompassed in the scope of the claims.

REFERENCES

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We claim:
 1. A method for suppressing the binding of one or moredetectable nucleic acid probes to one or more undesired sequences in anassay for determining target genomic nucleic acid of a sample; saidmethod comprising: a) contacting the sample with a mixture of two ormore different non-nucleotide probes wherein each probe is 16 to 50nucleobases in length and each probe comprises a nucleobase sequenceidentical to one of the nucleobase sequences of the group consisting of:Seq. Id. No. 3, Seq. Id. No. 4, Seq. Id. No. 5, Seq. Id. No. 6, Seq. Id.No. 7, Seq. Id. No. 8, Seq. Id. No. 9, Seq. Id. No. 10, Seq. Id. No. 11,Seq. Id. No. 12, Seq. Id. No. 13, and the complement thereof, whereinthe mixture of non-nucleotide probes further comprises at least oneprobe comprising a nucleobase sequence selected fromTT(k)TTTTT(k)TTTLysOLysOTTT(k)TTTTT(k)TT andAA(k)AAAAA(k)AAALysOLysOAAA(k)AAAAA(k)AA, wherein (k) is a D-lysine, Lysis L-lysine, and O is 8-amino-3,6-dioxaoctanoic acid; b) contacting thesample with the one or more detectable nucleic acid probes, wherein saidone or more detectable nucleic acid probes is 100 bp or greater, isderived from a genomic nucleic acid, and contains one or more segmentsof randomly distributed repeat sequence selected from the groupconsisting of SINEs and LINEs; and c) determining the target genomicnucleic acid of the sample by determining the hybridization of the oneor more detectable nucleic acid probes to the target genomic nucleicacid of the sample.
 2. The method of claim 1, wherein the two or morenon-nucleotide probes are peptide nucleic acid (PNA) oligomers.
 3. Themethod of claim 2, wherein the PNA subunits of the individual PNAoligomers have the formula:

wherein, each J is the same or different and is selected from the groupconsisting of: H, R¹, OR¹, SR¹, NHR¹, NR¹ ₂, F, Cl, Br and I; each K isthe same or different and is selected from the group consisting of: O,S, NH and NR¹; each R¹ is the same or different and is an alkyl grouphaving one to five carbon atoms which may optionally contain aheteroatom or a substituted or unsubstituted aryl group; each A isselected from the group consisting of a single bond, a group of theformula: —(CJ₂)_(s)- and a group of the formula; —(CJ₂)_(s)C(O)—wherein, J is defined above and each s is an integer from one to five;each t is 1 or 2; each u is 1 or 2; and each L is the same or differentand is independently selected from the group consisting of J, dabcyl,fluorescein, adenine, cytosine, guanine, thymine, uracil,5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine,2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine,pseudoisocytosine, 2-thiouracil, 2-thiothymidine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine),N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine), other naturally occurring nucleobaseanalogs, other non-naturally occurring nucleobases and substituted andunsubstituted aromatic moieties.
 4. The method of claim 3, wherein thePNA subunits of the individual PNA probes consist of a naturally ornon-naturally occurring nucleobase attached to an aza nitrogen of anN-[2-(aminoethyl)]glycine backbone through a methylene carbonyl linkage.5. The method of claim 1, wherein the mixture comprises 5 to about 50different non-nucleotide probes.
 6. The method of claim 5, wherein themixture comprises about 10 to about 25 different probes.
 7. The methodof claim 1, wherein the one or more detectable nucleic acid probes(s) islabeled with a fluorophore.